Amino acid modified polypeptides

ABSTRACT

Incorporation of certain amino acid analogs into polypeptides produced by cells which do not-ordinarily provide polypeptides containing such amino acid analogs is accomplished by subjecting the cells to growth media containing such amino acid analogs. The degree of incorporation can be regulated by adjusting the concentration of amino acid analogs in the media and/or by adjusting osmolality of the media. Such incorporation allows the chemical and physical characteristics of polypeptides to be altered and studied. In addition, nucleic acid and corresponding proteins including a domain from a physiologically active peptide and a domain from an extracellular matrix protein which is capable of providing a self-aggregate are provided. Human extracellular matrix proteins capable of providing a self-aggregate collagen are provided which are produced by prokaryotic cells. Preferred codon usage is employed to produce extracellular matrix proteins in prokaryotics.

BACKGROUND

1. Technical Field

Engineered polypeptides and chimeric polypeptides having incorporatedamino acids which enhance or otherwise modify properties of suchpolypeptides.

2. Description of Related Art

Genetic engineering allows polypeptide production to be transferred fromone organism to another. In doing so, a portion of the productionapparatus indigenous to an original host is transplanted into arecipient. Frequently, the original host has evolved certain uniqueprocessing pathways in association with polypeptide production which arenot contained in or transferred to the recipient. For example, it iswell known that mammalian cells incorporate a complex set ofpost-translational enzyme systems which impart unique characteristics toprotein products of the systems. When a gene encoding a protein normallyproduced by mammalian cells is transferred into a bacterial or yeastcell, the protein may not be subjected to such post translationalmodification and the protein may not function as originally intended.

Normally, the process of polypeptide or protein synthesis in livingcells involves transcription of DNA into RNA and translation of RNA intoprotein. Three forms of RNA are involved in protein synthesis: messengerRNA (mRNA) carries genetic information to ribosomes made of ribosomalRNA (rRNA) while transfer RNA (tRNA) links to free amino acids in thecell pool. Amino acid/tRNA complexes line up next to codons of mRNA,with actual recognition and binding being mediated by tRNA. Cells cancontain up to twenty amino acids which are combined and incorporated insequences of varying permutations into proteins. Each amino acid isdistinguished from the other nineteen amino acids and charged to tRNA byenzymes known as aminoacyl-tRNA synthetases. As a general rule, aminoacid/tRNA complexes are quite specific and normally only a molecule withan exact stereochemical configuration is acted upon by a particularaminoacyl-tRNA synthetase.

In many living cells some amino acids are taken up from the surroundingenvironment and some are synthesized within the cell from precursors,which in turn have been assimilated from outside the cell. In certaininstances, a cell is auxotrophic, i.e., it requires a specific growthsubstance beyond the minimum required for normal metabolism andreproduction which it must obtain from the surrounding environment. Someauxotrophs depend upon the external environment to supply certain aminoacids. This feature allows certain amino acid analogs to be incorporatedinto proteins produced by auxotrophs by taking advantage of relativelyrare exceptions to the above rule regarding stereochemical specificityof aminoacyl-tRNA synthetases. For example, proline is such anexception, i.e., the amino acid activating enzymes responsible for thesynthesis of prolyl-tRNA complex are not as specific as others. As aconsequence certain proline analogs have been incorporated intobacterial, plant, and animal cell systems. See Tan et al., ProlineAnalogues Inhibit Human Skin Fibroblast Growth and Collagen Productionin Culture, Journal of Investigative Dermatology, 80:261-267(1983).

A method of incorporating unnatural amino acids into proteins isdescribed, e.g., in Noren et al., A-General Method For Site-SpecificIncorporation of Unnatural Amino Acids Into Proteins, Science, Vol. 244,pp. 182-188 (1989) wherein chemically acylated suppressor tRNA is usedto insert an amino acid in response to a stop codon substituted for thecodon encoding residue of interest. See also, Dougherty et al.,Synthesis of a Genetically Engineered Repetitive Polypeptide ContainingPeriodic Selenomethionine Residues, Macromolecules, Vol. 26, No. 7, pp.1779-1781 (1993), which describes subjecting an E. coli methionineauxotroph to selenomethionine containing medium and postulates on thebasis of experimental data that selenomethionine may completely replacemethionine in all proteins produced by the cell.

cis-Hydroxy-L-proline has been used to study its effects on collagen byincorporation into eukaryotic cells such as cultured normal skinfibroblasts (see Tan et al., supra) and tendon cells from chick embryos(see e.g., Uitto et al., Procollagen Polypeptides Containingcis-4-Hydroxy-L-proline are Overglycosylated and Secreted as NonhelicalPro-γ-Chains, Archives of Biochemistry and Biophysics,185:1:214-221(1978)). However, investigators found thattrans-4-hydroxyproline would not link with proline specific tRNA ofprokaryotic E. coli. See Papas et al., Analysis of the Amino AcidBinding to the Proline Transfer Ribonucleic Acid Synthetase ofEscherichia coli, Journal of Biological Chemistry,245:7:1588-1595(1970). Another unsuccessful attempt to incorporatetrans-4-hydroxyproline into prokaryotes is described in Deming et al.,In Vitro Incorporation of Proline Analogs into Artificial Proteins,Poly. Mater. Sci. Engin. Proceed., Vol. 71, p. 673-674 (1994). Deming etal. report surveying the potential for incorporation of certain prolineanalogs, i.e., L-azetidine-2-carboxylic acid, L-γ-thiaproline,3,4-dehydroproline and L-trans-4-hydroxyproline into artificial proteinsexpressed in E. coli cells. Only L-azetidine-2-carboxylic acid,L-y-thiaproline and 3,4 dehydroproline are reported as beingincorporated into proteins in E. coli cells in vivo.

Extracellular matrix proteins (“EMPs”) are found in spaces around ornear cells of multicellular organisms and are typically fibrous proteinsof two functional types: mainly structural, e.g., collagen and elastin,and mainly adhesive, e.g., fibronectin and laminin. Collagens are afamily of fibrous proteins typically secreted by connective tissuecells. Twenty distinct collagen chains have been identified whichassemble to form a total of about ten different collagen molecules. Ageneral discussion of collagen is provided by Alberts, et al., The Cell,Garland Publishing, pp. 802-823 (1989), incorporated herein byreference. Other fibrous or filamentous proteins include Type I IFproteins, e.g., keratins; Type II IF proteins, e.g., vimentin, desminand glial fibrillary acidic protein; Type III IF proteins, e.g.,neurofilament proteins; and Type IV IF proteins, e.g., nuclear laminins.

Type I collagen is the most abundant form of the fibrillar, interstitialcollagens and is the main component of the extracellular matrix.Collagen monomers consist of about 1000 amino acid residues in arepeating array of Gly-X-Y triplets. Approximately 35% of the X and Ypositions are occupied by proline and trans 4-hydroxyproline. Collagenmonomers associate into triple helices which consist of one α2 and twoα1 chains. The triple helices associate into fibrils which are orientedinto tight bundles. The bundles of collagen fibrils are furtherorganized to form the scaffold for extracellular matrix.

In mammalian cells, post-translational modification of collagencontributes to its ultimate chemical and physical properties andincludes proteolytic digestion of pro-regions, hydroxylation of lysineand proline, and glycosylation of hydroxylated lysine. The proteolyticdigestion of collagen involves the cleavage of pro regions from the Nand C termini. It is known that hydroxylation of proline is essentialfor the mechanical properties of collagen. Collagen with low levels of4-hydroxyproline has poor mechanical properties, as highlighted by thesequelae associated with scurvy. 4-hydroxyproline adds stability to thetriple helix through hydrogen bonding and through restricting rotationabout C—N bonds in the polypeptide backbone. In the absence of a stablestructure, naturally occurring cellular enzymes contribute to degradingthe collagen polypeptide.

The structural attributes of Type I collagen along with its generallyperceived biocompatability make it a desirable surgical implantmaterial. Collagen is purified from bovine skin or tendon and used tofashion a variety of medical devices including hemostats, implantablegels, drug delivery vehicles and bone substitutes. However, whenimplanted into humans bovine collagen can cause acute and delayed immuneresponses.

As a consequence, researchers have attempted to produce humanrecombinant collagen with all of its structural attributes in commercialquantities through genetic engineering. Unfortunately, production ofcollagen by commercial mass producers of protein such as E. coli has notbeen successful. A major problem is the extensive post-translationalmodification of collagen by enzymes not present in E. coli. Failure ofE. coli cells to provide proline hydroxylation of unhydroxylatedcollagen proline prevents manufacture of structurally sound collagen incommercial quantities.

Another problem in attempting to use E. coli to produce human collagenis that E. coli prefer particular codons in the production ofpolypeptides. Although the genetic code is identical in both prokaryoticand eukaryotic organisms, the particular codon (of the several possiblefor most amino acids) that is most commonly utilized can vary widelybetween prokaryotes and eukaryotes. See, Wada, K.-N., Y. Wada, F.Ishibashi, T. Gojobori and T. Ikemura. Nucleic Acids Res. 20,Supplement: 2111-2118, 1992. Efficient expression of heterologous (e.g.mammalian) genes in prokaryotes such as E. coli can be adverselyaffected by the presence in the gene of codons infrequently used in E.coli and expression levels of the heterologous protein often rise whenrare codons are replaced by more common ones. See, e.g., Williams, D.P., D. Regier, D. Akiyoshi, F. Genbauffe and J. R. Murphy. Nucleic AcidsRes. 16: 10453-10467, 1988 and Höög, J.-O., H. v. Bahr-Lindström, H.Jömvall and A. Holmgren. Gene. 43: 13-21, 1986. This phenomenon isthought to be related, at least in part, to the observation that a lowfrequency of occurrence of a particular codon correlates with a lowcellular level of the transfer RNA for that codon. See, Ikemura, T. J.Mol. Biol. 158: 573-597, 1982 and Ikemura, T. J. Mol. Biol. 146: 1-21,1981. Thus, the cellular tRNA level may limit the rate of translation ofthe codon and therefore influence the overall translation rate of thefull-length protein. See, Ikemura, T. J. Mol. Biol. 146: 1-21, 1981;Bonekamp, F. and F. K. Jensen. Nucleic Acids Res. 16: 3013-3024, 1988;Misra, R. and P. Reeves, Eur. J. Biochem. 152: 151-155, 1985; and Post,L. E., G. D. Strycharz, M. Nomura, H. Lewis and P. P. Lewis. Proc. Natl.Acad. Sci. U.S.A. 76: 1697-1701, 1979. In support of this hypothesis isthe observation that the genes for abundant E. coli proteins generallyexhibit bias towards commonly used codons that represent highly abundanttRNAs. See, Ikemura, T. J. Mol. Biol. 146: 1-21, 1981; Bonekamp, F. andF. K. Jensen. Nucleic Acids Res. 16: 3013-3024, 1988; Misra, R. and P.Reeves, Eur. J. Biochem. 152: 151-155, 1985; and Post, L. E., G. D.Strycharz, M. Nomura, H. Lewis and P. P. Lewis. Proc. Natl. Acad. Sci.U.S.A. 76: 1697-1701, 1979. In addition to codon frequency, the codoncontext (i.e. the surrounding nucleotides) can also affect expression.

Although it would appear that substituting preferred codons for rarecodons could be expected to increase expression of heterologous proteinsin host organisms, such is not the case. Indeed, “it has not beenpossible to formulate general and unambiguous rules to predict whetherthe content of low-usage codons in a specific gene might adverselyaffect the efficiency of its expression in E. coli.” See page 524 of S.C. Makrides (1996), Strategies for Achieving High-Level Expression ofGenes in Escherichia coli. Microbiological Reviews 60, 5.12-538. Forexample, in one case, various gene fusions between yeast α factor andsomatomedin C were made that differed only in coding sequence. In theseexperiments, no correlation was found between codon bias and expressionlevels in E. coli. Ernst, J. F. and Kawashima, E. (1988), J.Biotechnology, 7, 1-10. In another instance, it was shown that despitethe higher frequency of optimal codons in a synthetic β-globin genecompared to the native sequence, no difference was found in the proteinexpression from these two constructs when they were placed behind the T7promoter. Heman et al. (1992), Biochemistry, 31, 8619-8628. Conversely,there are many examples of proteins with a relatively high percentage ofrare codons that are well expressed in E. coli. A table listing some ofthese examples and a general discussion can be found in Makoff, A. J. etal. (1989), Nucleic Acids Research, 17, 10191-10202. In one case,introduction of non-optimal, rare arginine codons at the 3′ end of agene actually increased the yield of expressed protein. Gursky, Y. G.and Beabealashvilli, R. Sh. (1994), Gene 148, 15-21.

Failure to provide post-translational modifications such ashydroxylation of proline and the presence in human collagen of rarecodons for E. coli may be contributing to the difficulties encounteredin the expression of human collagen genes in E. coli.

SUMMARY

A method of incorporating an amino acid analog into a polypeptideproduced by a cell is provided which includes providing a cell selectedfrom the group consisting of prokaryotic cell and eukaryotic cell,providing growth media containing at least one amino acid analogselected from the group consisting of trans-4-hydroxyproline,3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof andcontacting the cell with the growth media wherein the at least one aminoacid analog is assimilated into the cell and incorporated into at leastone polypeptide.

Also provided is a method of substituting an amino acid analog of anamino acid in a polypeptide produced by a cell selected from the groupconsisting of prokaryotic cell and eukaryotic cell, which includesproviding a cell selected from the group consisting of prokaryotic celland eukaryotic cell, providing growth media containing at least oneamino acid analog selected from the group consisting oftrans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline andcombinations thereof and contacting the cell with the growth mediawherein the at least one amino acid analog is assimilated into the celland incorporated as a substitution for at least one naturally occurringamino acid in at least one polypeptide.

A method of controlling the amount of an amino acid analog incorporatedinto a polypeptide is also provided which includes providing at least afirst cell selected from the group consisting of prokaryotic cell andeukaryotic cell, providing a first growth media containing a firstpredetermined amount of at least one amino acid analog selected from thegroup consisting of trans-4-hydroxyproline, 3-hydroxyproline,cis-4-fluoro-L-proline and combinations thereof and contacting the firstcell with the first growth media wherein a first amount of amino acidanalog is assimilated into the first cell and incorporated into at leastone polypeptide. At least a second cell selected from the groupconsisting of prokaryotic cell and eukaryotic cell, is also providedalong with a second growth media containing a second predeterminedamount of an amino acid analog selected from the group consisting oftrans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline andcombinations thereof and the at least second cell is contacted with thesecond growth media wherein a second amount of amino acid analog isassimilated into the second cell and incorporated into at least onepolypeptide.

Also provided is a method of increasing stability of a recombinantpolypeptide produced by a cell which includes providing a cell selectedfrom the group consisting of prokaryotic cell and eukaryotic cell, andproviding growth media containing an amino acid analog selected from thegroup consisting of trans-4-hydroxyproline, 3-hydroxyproline,cis-4-fluoro-L-proline and combinations thereof and contacting the cellwith the growth media wherein the amino acid analog is assimilated intothe cell and incorporated into a recombinant polypeptide, therebystabilizing the polypeptide.

A method of increasing uptake of an amino acid analog into a cell andcausing formation of an amino acid analog/tRNA complex is also providedwhich includes providing a cell selected from the group consisting ofprokaryotic cell and eukaryotic cell, providing hypertonic growth mediacontaining amino acid analog selected from the group consisting oftrans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline andcombinations thereof and contacting the cell with the hypertonic growthmedia wherein the amino acid analog is assimilated into the cell andincorporated into an amino acid analog/tRNA complex. In any of the otherabove methods, a hypertonic growth media can optionally be incorporatedto increase uptake of an amino-acid analog into a cell.

A composition is provided which includes a cell selected from the groupconsisting of prokaryotic cell and eukaryotic cell, and hypertonic mediaincluding an amino acid analog selected from the group consisting oftrans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline andcombinations thereof.

Also provided is a method of producing an Extracellular Matrix Protein(EMP) or a fragment thereof capable of providing a self-aggregate in acell which does not ordinarily hydroxylate proline which includesproviding a nucleic acid sequence encoding the EMP or fragment thereofwhich has been optimized for expression in the cell by substitution ofcodons preferred by the cell for naturally occurring codons notpreferred by the cell, incorporating the nucleic acid sequence into thecell, providing hypertonic growth media containing at least one aminoacid selected from the group consisting of trans-4-hydroxyproline and3-hydroxyproline, and contacting the cell with the growth media whereinthe at least one amino acid is assimilated into the cell andincorporated into the EMP or fragment thereof.

Nucleic acid encoding a chimeric protein is provided which includes adomain from a physiologically active peptide and a domain from anextracellular matrix protein (EMP) which is capable of providing aself-aggregate. The nucleic acid may be inserted into a cloning vectorwhich can then be incorporated into a cell.

Also provided is a chimeric protein including a domain from aphysiologically active peptide and a domain from an extracellular matrixprotein (EMP) which is capable of providing a self aggregate.

Also provided is human collagen produced by a prokaryotic cell, thehuman collagen being capable of providing a self aggregate.

Also provided is nucleic acid encoding a human Extracellular MatrixProtein (EMP) wherein the codon usage in the nucleic acid sequencereflects preferred codon usage in a prokaryotic cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plasmid map illustrating pMAL-c2.

FIG. 2 is a graphical representation of the concentration ofintracellular hydroxyproline based upon concentration oftrans-4-hydroxyproline in growth culture over time.

FIG. 2A is a graphical representation of the concentration ofintracellular hydroxyproline as a function of sodium chlorideconcentration.

FIGS. 3A and 3B depict a DNA sequence encoding human Type 1 (α₁)collagen (SEQ. ID. NO. 1).

FIG. 4 is a plasmid map illustrating pHuCol.

FIG. 5 depicts a DNA sequence encoding a fragment of human Type 1 (α₁)collagen (SEQ. ID. NO; 2).

FIG. 6 is a plasmid map illustrating pHuCol-Fl.

FIG. 7 depicts a DNA sequence encoding a collagen-like peptide whereinthe region coding for gene collagen-like peptide is underlined (SEQ. ID.NO. 3).

FIG. 8 depicts an amino acid sequence of a collagen-like peptide (SEQ.ID. NO. 4).

FIG. 9 is a plasmid map illustrating pCLP.

FIG. 10 depicts a DNA sequence encoding mature bone morphogenic protein(SEQ. ID. NO. 5).

FIG. 11 is a plasmid map illustrating pCBC.

FIG. 12 is a graphical representation of the percent incorporation ofproline and trans-4-hydroxyproline into maltose binding protein undervarious conditions.

FIG. 13 depicts a collagen I (α1)/BMP-2B chimeric amino acid sequence(SEQ. ID. NO. 6).

FIG. 14A-14C depicts a collagen I (α1)/BMP-2B chimeric nucleotidesequence (SEQ. ID. NO. 7).

FIG. 15 depicts a collagen I (α1)/TGF-plamino acid sequence (SEQ. ID.NO. 8).

FIG. 16A-16C depict a collagen I (α1)/TGF-β₁ nucleotide sequence (SEQ.ID. NO. 9). Lower case lettering indicates non-coding sequence.

FIGS. 17A-17B depict a collagen I (α₁)/decorin amino acid sequence (SEQ.ID. NO. 10).

FIG. 18 depicts a collagen I (α1)/decorin peptide amino acid sequence(SEQ. ID. NO. 11).

FIGS. 19A-19D depict a collagen I (α1)/decorin nucleotide sequence (SEQ.ID. NO. 12).

FIGS. 20A-20C depict a collagen/decorin peptide nucleotide sequence(SEQ. ID. NO. 13). Lower case lettering indicates non-coding sequence.

FIG. 21 depicts a pMal cloning vector and polylinker cloning site.

FIG. 22 depicts a polylinker cloning site contained in the pMal cloningvector of FIG. 21 (SEQ. ID. NO. 14).

FIG. 23 depicts a pMal cloning vector containing a BMP/collagennucleotide chimeric construct.

FIG. 24 depicts a pMal cloning vector containing a TGF-β₁/collagennucleotide chimeric construct.

FIG. 25 depicts a pMal cloning vector containing a decorin/collagennucleotide chimeric construct.

FIG. 26 depicts a pMal cloning vector containing a decorinpeptide/collagen nucleotide chimeric construct.

FIG. 27A-27E depicts a human collagen Type I (α₁) nucleotide sequence(SEQ. ID. NO. 15) and corresponding amino acid sequence (SEQ. ID. NO.16).

FIG. 28 is a schematic diagram of the construction of the human collagengene from synthetic oligonucleotides.

FIG. 29 is a schematic depiction of the amino acid sequence of chimericproteins GST-ColECol (SEQ. ID. NO. 17) and GST-D4 (SEQ. ID. NO. 18).

FIG. 30 is a Table depicting occurrence of four proline and four glycinecodons in the human Collagen Type I (α₁) gene with optimized codon usage(ColECol).

FIG. 31 depicts a gel reflecting expression and dependence of expressionof GST-D4 on hydroxyproline.

FIG. 32 depicts a gel showing expression of GST-D4 in hypertonic media

FIG. 33 is a graph showing circular dichroism spectra of native anddenatured D4 in neutral phosphate buffer.

FIG. 34 depicts a gel representing digestion of D4 with bovine pepsin..

FIG. 35 depicts a gel representing expression of GST-H Col andGST-ColECol under specified conditions.

FIG. 36 depicts a gel representing expression of GST-CM4 in media withor without NaCl and either proline or hydroxyproline.

FIG. 37 depicts a gel of six hour post induction samples of GST-CM4expressed in E. coli with varying concentrations of NaCl.

FIG. 38 depicts a gel of 4 hour post induction samples of GST-CM4expressed in E. coli with constant amounts of hydroxyproline and varyingamounts of proline.

FIGS. 39A-39E depict the nucleotide (SEQ. ID. NO. 19) and amino acid(SEQ. ID. NO. 20) sequence of HuColE, the helical region of human Type I(α₁) collagen plus 17 amino terminal extra-helical amino acids and 26carboxy terminal extra-helical amino acids with codon usage optimizedfor E. coli.

FIG. 40 depicts sequence and restriction maps of synthetic oligos usedto reconstruct the first 243 base pairs of the human Type I (α₁)collagen gene with optimized E. coli codon usage. The synthetic oligosare labelled N1-1 (SEQ. ID. NO. 21), N1-2 (SEQ. ID. NO. 22), N1-3 (SEQ.ID. NO. 23) and N1-4 (SEQ. ID. NO. 24).

FIG. 41 depicts a plasmid map of pBSN1-1 containing a 114 base pairfragment of human collagen Type I (α₁) with optimized E. coli codonusage.

FIG. 42 depicts the nucleotide (SEQ. ID. NO. 25) and amino acid (SEQ.ID. NO. 26) sequence of a fragment of human collagen Type I (α₁) genewith optimized E. coli codon usage encoded by plasmid pBSNI-1.

FIG. 43 depicts a plasmid map of pBSNl-2 containing a 243 base pairfragment of human collagen Type I (α₁) with optimized E. coli codonusage.

FIG. 44 depicts the nucleotide (SEQ. ID. NO. 27) and amino acid (SEQ.ID. NO. 28) sequence of a fragment of human collagen Type I (α₁) genewith optimized E. coli codon usage encoded by plasmid pBSN1-2.

FIG. 45 depicts a plasmid map of pHuCol^(Ec) containing human collagenType I (α₁) with optimized E. coli codon usage.

FIG. 46 depicts a plasmid map of pTrc N1-2 containing a 234 nucleotide.human collagen Type I (α₁) fragment with optimized E. coli codon usage.

FIG. 47 depicts a plasmid map of pNI-3 containing a 360 nucleotide humancollagen Type I (α₁) fragment with optimized E. coli codon usage.

FIG. 48 depicts a plasmid map of pD4 containing a 657 nucleotide humancollagen Type I (α₁) 3′ fragment with optimized E. coli codon usage.

FIGS. 49A-49E depict the nucleotide (SEQ. ID. NO. 29) and amino acid(SEQ. ID. NO. 30) sequence of a helical region of human Type I (α₂)collagen plus 11 amino terminal extra-helical amino acids and 12 carboxyterminal extrahelical amino acids.

FIGS. 50A-50E depict the nucleotide (SEQ. ID. NO. 31) and amino acid(SEQ. ID. NO. 32) sequence of HuCol(α₂)^(Ec), the helical region ofhuman Type I (α₂) collagen plus 11 amino terminal extra-helical aminoacids and 12 carboxy terminal extra-helical amino acids with codon usageoptimized for E. coli.

FIG. 51 depicts sequence and restriction maps of synthetic oligos usedto reconstruct the first 240 base pairs of human Type I (α₂) collagengene with optimized E. coli codon usage. The synthetic oligos arelabelled N1-1 (α₂) (SEQ. ID. NO. 33), N1-2 (α2) (SEQ. ID. NO. 34), N1-3(α2) (SEQ. ID. NO. 35) and N1-4 (α2) (SEQ. ID. NO. 36).

FIG. 52 depicts a plasmid map of pBSN1-1 (α₂) containing a 117 base pairfragment of human collagen Type I (α₂) with optimized E. coli codonusage.

FIG. 53 depicts a plasmid map of pBSN1-2 (α₂) containing a 240 base pairfragment of human collagen Type I (α₂) with optimized E. coli codonusage.

FIG. 54 depicts the nucleotide (SEQ. ID. NO. 37) and amino acid (SEQ.ID. NO. 38) sequence of a fragment of human collagen Type I (α₂) genewith optimized E. coli usage encoded by plasmid pBSN1-2(α₂).

FIG. 55 depicts a plasmid map of pHuCol(α₂)^(Ec) containing the entire.human collagen Type I (α₂) gene with optimized E. coli codon usage.

FIG. 56 depicts a plasmid map of pN1-2 (α₂) containing a 240 base pairfragment of human collagen Type I (α₂) with optimized E. coli codonusage.

FIG. 57 depicts a gel reflecting expression of GST and TGF-β1 underspecified conditions.

FIG. 58 depicts a gel reflecting expression of MBP, FN-BMP-2A, FN-TGF-β1and FN under specified conditions.

FIG. 59 depicts a gel showing expression of GST-Coll under specifiedconditions.

FIG. 60 depicts a plasmid map of pGST-CM4 containing the gene forglutathione S-transferase fused to the gene for collagen mimetic 4.

FIG. 61 depicts the nucleotide (SEQ. ID. NO. 39) and amino acid (SEQ.ID. NO. 40) sequence of collagen mimetic 4.

FIG. 62A depicts a chromatogram of the elution of hydroxyprolinecontaining collagen mimetic 4 from a Poros RP2 colurnr. The arrowindicates the peak containing hydroxyproline containing collagen mimetic4.

FIG. 62B depicts a chromatogram of the elution of proline-containingcollagen mimetic 4 from a Poros RP2 column. The arrow indicates the peakcontaining proline containing collagen mimetic 4.

FIG. 63A depicts a chromatogram of a proline amino acid standard (250pmol).

FIG. 63B depicts a chromatogram of a hydroxyproline amino acid standard(250 pmol).

FIG. 63C depicts an amino acid analysis chromatogram of the hydrolysisof proline containing collagen mimetic 4.

FIG. 63D depicts an amino acid analysis chromatogram of the hydrolysisof hydroxyproline containing collagen mimetic 4.

FIG. 64 is a graph of OD600 versus time for cultures of E. coli JM109(F—) grown to plateau and then supplemented with various amino acids.

FIG. 65 depicts a plasmid map of pcEc-α1 containing the gene forHuCol(α1)E^(Ec).

FIG. 66 depicts a plasrnid map of pcEc-α2 containing the gene forHuCol(α2)^(Ec).

FIG. 67 depicts a plasmid map of pD4-α1 containing the gene for a 219amino acid C-terminal fragment of Type I (α1) human collagen withoptimized E. coli codon usage fused to the gene for glutathioneS-transferase.

FIG. 68 depicts a plasmid map of pD4-α2 containing the gene for a 207arnino acid C-terminal fragment of Type I (α2) human collagen withoptimized E. coli codon usage fused to the gene for glutathioneS-tranrferase.

FIG. 69 depicts the predicted amino acid sequence from the DNA sequenceof the first 13 amino acid acids of protein D4-α1 (SEQ. ID. NO. 41) andthe amino acid sequence as experimentally determined (SEQ. ID NO. 42).

FIG. 70 depicts the mass spectrum of hydroxyproline containing D4-α1.

FIG. 71 depicts the nucleotide sequence of a 657 nucleotide humancollagen Type I (α1)3′ fragment with optimized E. coli codon usagedesignated D4 (SEQ. ID.NO. 43).

FIG. 72 depicts the amino acid sequence of a 219 amino acid C-terminalfragment of humran collagen Type I (α1) designed D4 (SEQ. ID. NO. 44).

FIG. 73 is a plasmid map illustrating pGEX-4T.1 containing the gene forglutatione S-transferase.

FIG. 74 is a plasmid map illustrating pTrc-TGF containing the gene forthe mature human TGF-β1 polypeptide.

FIG. 75 is a plasmid map illustrating pTrc-Fn containing the gene for a70 kDa fragment of human fibronectin.

FIG. 76 is a plasmid map illustrating pTrc-Fn-TGF containing the genefor a fusion protein of a 70 kDA fragment of human fibronectin and themature human TGF-β1 polypeptide.

FIG. 77 is a plasmid map illustrating pTrc-Fn-BMP containing the genefor a fusion protein of a 70 kDa fragment of human fibronectin and humanbone morphogenic protein 2A.

FIG. 78 is a plasmid map illustrating pGEX-HuColl^(Ec) containing thegene for a fusion between glutathione S-transferase and Type I (α1)human collagen with optimized E. coli codon usage.

FIG. 79 depicts the nucleotide sequence of a 627 nucleotide humancollagen Type I (α2) 3′ fragment with optimized E. coli codon usage(SEQ. ID. NO.45).

FIG. 80 depicts the amino acid sequence of a 209 amino acid C-terminalfragment of human collagen Type I (α2) (SEQ. ID.. NO. 46).

FIG. 81 depicts the sequence of synthetic oligos used to reconstruct thefirst 282 base pairs of the gene for the carboxy terminal 219 aminoacids of human. Type I (α1) collagen with optimized E. coli-codon usagedesignated N4-1 (SEQ. ID. NO. 47), N4-2 (SEQ. ID. NO. 48), N4-3 (SEQ.ID. NO. 49) and N4-4 (SEQ. ID. NO. 50).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Prokaryotic cells and eukaryotic cells can unexpectedly be made toassimilate and incorporate trans-4-hydroxyproline into proteins contraryto both Papas et al. and Deming et al., supra. Such assimilation andincorporation is especially useful when the structure and function of apolypeptide depends on post translational hydroxylation of proline notprovided by the native protein production system of a recombinant host.Thus, prokaryotic bacteria such as E. coli and eukaryotic cells such asSaccharomyces cerevisiae, Saccharomyces carlsbergensis andSchizosaccharomyces pombe that ordinarily do not hydroxylate proline andadditional eukaryotes such as insect cells including lepidopteran celllines including Spodoptera frugiperda, Trichoplasia ni, Heliothisvirescens, Bombyx mori infected with a baculovirus; CHO cells, COS cellsand NIH 3T3 cells which fail to adequately produce certain polypeptideswhose structure and function depend on such hydroxylation can be made toproduce polypeptides having hydroxylated prolines. Incorporationincludes adding trans-4-hydroxyproline to a polypeptide, for example, byfirst changing an amino acid to proline, creating a new proline positionthat can in turn be substituted with trans4-hydroxyproline orsubstituting a naturally occurring proline in a polypeptide withtrans-4-hydroxyproline as well.

The process of producing recombinant polypeptides in mass producingorganisms is well known. Replicable expression vectors such as plasmids,viruses, cosmids and artificial chromosomes are cornmonly used totransport genes encoding desired proteins from one host to another. Itis contemplated that any known method of cloning a gene, ligating thegene into an expression vector and transforming a host cell with suchexpression vector can be used in furtherance of the present disclosure.

Not only is incorporation of trans-4-hydroxyproline into polypeptideswhich depend upon trans-4-hydroxyproline for chemical and physicalproperties useful in production systems which do not have theappropriate systems for converting proline to trans-4-hydroxyproline,but useful as well in studying the structure and function ofpolypeptides which do not normally contain trans-4-hydroxyproline. It iscontemplated that the following amino acid analogs may also beincorporated in accordance with the present disclosure: trans-4hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline andcombinations thereof (hereinafter referred to as the “amino acidanalogs”). Use of prokaryotes and eukaryotes is desirable since theyallow relatively inexpensive mass production of such polypeptides. It iscontemplated that the amino acid analogs can be incorporated into anydesired polypeptide. In a preferred embodiment the prokaryotic cells andeukaryotic cells are starved for proline by decreasing or eliminatingthe amount of proline in growth media prior to addition of an amino acidanalog herein.

Expression vectors containing the gene for maltose binding protein(MBP), e.g., see FIG. 1 illustrating plasmid pMAL-c2, commerciallyavailable from New England Bio-Labs, are transformed into prokaryotessuch as E. coli proline auxotrophs or eukaryotes such as S. cerevisiaeauxotrophs which depend upon externally supplied proline for proteinsynthesis and anabolism. Other preferred expression vectors for use inprokaryotes are commercially available plasmids which include pKK-223(Pharmacia), pTRC (Invitrogen), pGEX (Pharmacia), pET (Novagen) and pQE(Quiagen). It should be understood that any suitable expression vectormay be utilized by those with skill in the art.

Substitution of the amino acid analogs for proline in protein synthesisoccurs since prolyl tRNA synthetase is sufficiently promiscuous to allowmisacylation of proline tRNA with any one of the amino acid analogs. Asufficient quantity, i.e., typically ranging from about 0.001M to about1.0 M, but more preferably from about 0.005M to about 0.5M of the aminoacid analog(s) is added to the growth medium for the transformed cellsto compete with proline in cellular uptake. After sufficient time,generally from about 30 minutes to about 24 hours or more, the aminoacid analog(s) is assimilated by the cell and incorporated into proteinsynthetic pathways. As can be seen from FIGS. 2 and 2A, intracellularconcentration of trans-4-hydroxyproline increases by increasing theconcentration of sodium chloride in the growth media. In a preferredembodiment the prokaryotic cells and/or eukaryotic cells are starved forproline by decreasing or eliminating the amount of proline in growthmedia prior to addition of an amino acid analog herein.

Expression vectors containing the gene for human Type I (α1) collagen(DNA sequence illustrated in FIGS. 3 and 3A; plasmid map illustrated inFIG. 4) are transformed into prokaryotic or eukaryotic prolineauxotrophs which depend upon externally supplied proline for proteinsynthesis and anabolism. As above, substitution of the amino acidanalog(s) occurs since prolyl tRNA synthetase is sufficientlypromiscuous to allow misacylation of proline tRNA with the amino acidanalog(s). The quantity of amino acid analog(s) in media given above isagain applicable.

Expression vectors containing DNA encoding fragments of human Type I(α1) collagen (e.g., DNA sequence illustrated in FIG. 5 and plasmid mapillustrated in FIG. 6) are transformed into prokaryotic or eukaryoticauxotrophs as above. Likewise, expression vectors containing DNAencoding collagen-like polypeptide (e.g., DNA sequence illustrated inFIG. 7, amino acid sequence illustration in FIG. 8 and plasmid mapillustrated in FIG. 9) can be used to transform prokaryotic oreukaryotic auxotrophs as above. Collagen-like peptides are those whichcontain at least partial homology with cohagen and exhibit similarchemical and physical characteristics to collagen. Thus, collagen-likepeptides consist, e.g., of repeating arrays of Gly-X-Y triplets in whichabout 35% of the X and Y positions are occupied by proline and4-hydroxyproline. Collagen-like peptides are interchangeably referred toherein as collagen-like proteins, collagen-like polypeptides, collagenmimetic polypeptides and collagen mimetic. Certain preferred collagenfragments and collagen-like peptides in accordance herewith are capableof assembling into an extracellular matrix. In both collagen fragmentsand collagen-like peptides as described above, substitution with aminoacid analog(s) occurs since prolyl tRNA synthetase is sufficientlypromiscuous to allow misacylation of proline tRNA with one or more ofthe amino acid analog(s). The quantity of amino acid analog(s) givenabove is again applicable.

It is contemplated that any polypeptide having an extracellular matrixprotein domain such as a collagen, collagen fragment or collagen-likepeptide domain can be made to incorporate amino acid analog(s) inaccordance with the disclosure herein. Such polypeptides includecollagen, a collagen fragment or collagen-like peptide domain and adomain having a region incorporating one or more physiologically activeagents such as glycoproteins, proteins, peptides and proteoglycans. Asused herein, physiologically active agents exert control over or modifyexisting physiologic functions in living things. Physiologically activeagents include hormones, growth factors, enzymes, ligands and receptors.Many active domains of physiologically active agents have been definedand isolated. It is contemplated that polypeptides having a collagen,collagen fragment or collagen-like peptide domain can also have a domainincorporating one or more physiologically active domains which areactive fragments of such physiologically active agents. As used herein,physiologically active agent is meant to include entire peptides,polypeptides, proteins, glycoproteins, proteoglycans and activefragments of any of them. Thus, chimeric proteins are made toincorporate amino acid analog(s) by transforming a prokaryotic prolineauxotroph or a eukaryotic proline auxotroph with an appropriateexpression vector and contacting the transformed - auxotroph with growthmedia containing at least one of the amino acid analogs. For example, achimeric collagen/bone morphogenic protein (BMP) construct or variouschimeric collagen/growth factor constructs are useful in accordanceherein. Such growth factors are well-known and include insulin-likegrowth factor, transforming growth factor, platelet derived growthfactor and the like. FIG. 10 illustrates DNA of BMP which can be fusedto the 3′ terminus of DNA encoding collagen, DNA encoding a collagenfragment or DNA encoding a collagen-like peptide. FIG. 11 illustrates amap of plasmid pCBC containing a collagen/BMP construct. In a preferredembodiment, proteins having a collagen, collagen fragment orcollagen-like peptide domain assemble or aggregate to form anextracellular matrix which can be used as a surgical implant. Theproperty of self-aggregation as used herein includes the ability to forman aggregate with the same or similar molecules or to form an aggregatewith different molecules that share the property of aggregation to form,e.g., a double or triple helix. An example of such aggregation is thestructure of assembled collagen matrices.

Indeed, chimeric polypeptides which may also be referred to herein aschimericproteins provide an integrated combination of a therapeuticallyactive domain from a physiologically active agent and one or more EMPmoieties. The EMP domain provides an integral vehicle for delivery ofthe therapeutically active moiety to a target site. The two domains arelinked covalently by one or more peptide bonds contained in a linkerregion. As used herein, integrated or integral means characteristicswhich result from the covalent association of one or more domains of thechimeric proteins. The therapeutically active moieties disclosed hereinare typically made of amino acids linked to form peptides, polypeptides,proteins, glycoproteins or proteoglycans. As used herein, peptideencompasses polypeptides and proteins.

The inherent characteristics of EMPs are ideal for use as a vehicle forthe therapeutic moiety. One such characteristic is the ability of theEMPs to form the self-aggregate. Examples of suitable EMPs are collagen,elastin, fibronectin, fibrinogen and fibrin. Fibrillar collagens (TypeI, II and III) assemble into ordered polymers and often aggregate intolarger bundles. Type IV collagen assembles into sheetlike meshworks.Elastin molecules form filaments and sheets in which the elastinmolecules are highly cross-linked to one another to provide goodelasticity and high tensile strength. The cross-linked, random-coiledstructure of the fiber network allows it to stretch and recoil like arubber band. Fibronectin is a large fibril forming glycoprotein, which,in one of its forms, consists of highly insoluble fibrils cross-linkedto each other by disulfide bonds. Fibrin is an insoluble protein formedfrom fibrinogen by the proteolytic activity of thrombin during thenormal clotting of blood.

The molecular and macromolecular morphology of the above EMPs definesnetworks or matrices to provide substratum or scaffolding in integralcovalent association with the therapeutically active moiety. Thenetworks or matrices formed by the EMP domain provide an environmentparticularly well suited for ingrowth of autologous cells involved ingrowth, repair and replacement of existing tissue. The integraltherapeutically active moieties covalently bound within the networks ormatrices provide maximum exposure of the active agents to their targetsto elicit a desired response.

Implants formed of or from the present chimeric proteins providesustained release activity in or at a desired locus or target site.Since it is linked to an EMP domain, the therapeutically active domainof the present chimeric protein is not free to separately diffuse orotherwise be transported away from the vehicle which carries it, absentcleavage of peptide bonds. Consequently, chimeric proteins hereinprovide an effective anchor for therapeutic activity which allows theactivity to be confined to a target location for a prolonged duration.Because the supply of therapeutically active agent does not have to bereplenished as often when compared to non-sustained release dosageforms, smaller amounts of therapeutically active agent may be used overthe course of therapy. Consequently, certain advantages provided by thepresent chimeric proteins are a decrease or elimination of local andsystemic side effects, less potentiation or reduction in therapeuticactivity with chronic use, and minimization of drug accumulation in bodytissue with chronic dosing.

Use of recombinant technology allows manufacturing of non-immunogenicchimeric proteins. The DNA encoding both the therapeutically activemoiety and the EMP moiety should preferably be derived from the samespecies as the patient being treated to avoid an immunogenic reaction.For example, if the patient is human, the therapeutically active moietyas well as the EMP moiety is preferably derived from human DNA.

Osteogenic/EMP chimeric proteins provide biodegradable and biocompatibleagents for inducing bone formation at a desired site. As stated above,in one embodiment, a BMP moiety is covalently linked with an EMP to formchimeric protein. The BMP moiety induces osteogenesis and theextracellular matrix protein moiety provides an integral substratum orscaffolding for the BMP moiety and cells which are involved inreconstruction and growth. Compositions containing the BMP/EMP chimericprotein provide effective sustained release delivery of the BMP moietyto desired target sites. The method of manufacturing such an osteogenicagent is efficient because the need for extra time consuming steps aspurifying EMP and then admixing it with the purified BMP are eliminated.An added advantage of the BMP/EMP chimeric protein results from thestability created by the covalent bond between BMP and the EMP, i.e.,the BMP portion is not free to separately diffluse away from the EMP,thus providing a more stable therapeutic agent.

Bone morphogenic proteins are class identified as BMP-1 through BMP-9. Apreferred osteogenic protein for use in human patients is human BMP-2B.A BMP-2B/collagen IA chimeric protein is illustrated in FIG. 13 (SEQ.ID. NO. 6). The protein sequence illustrated in FIG. 15 (SEQ. ID. NO. 8)includes a collagen helical domain depicted at amino acids 1-1057 and amature form of BMP-2B at amino acids 1060-1169. The physical propertiesof the chimeric protein are dominated in part by the EMP component. Inthe case of a collagen moiety, a concentrated solution of chimericprotein will have a gelatinous consistency that allows easy handling bythe medical practitioner. The EMP moiety acts as a sequestering agent toprevent rapid desorption of the BMP moiety from the desired site and toprovide sustained release of BMP activity. As a result, the BMP moietyremains at the desired site and provides sustained release of BMPactivity at the desired-site for a period of time necessary toeffectively induce bone formation. The EMP moiety also provides a matrixwhich allows a patient's autologous cells, e.g., chondrocytes and thelike, which are normally involved in osteogenesis to collect therein andform an autologous network for new tissue growth. The gelatinousconsistency of the chimeric protein also provides a useful andconvenient therapeutic manner for immobilizing active BMP on a suitablevehicle or implant for delivering the BMP moiety to a site where bonegrowth is desired.

The BMP moiety and the EMP moiety are optionally linked together bylinker sequences of amino acids. Examples of linker sequences used areillustrated within the sequence depicted in FIGS. 14A-14C (SEQ. ID. NO.7), 16A-16C (SEQ. ID. NO. 9), 19A-19C (SEQ. ID. NO. 12) and 20A-20C(SEQ. ID. NO. 13), and are described in more detail below. Linkersequences may be chosen based on particular properties which they impartto the chimeric protein. For example, amino acid sequences such asIle-Glu-Gly Arg and Leu-Val-Pro-Arg are cleaved by factor XA andthrombin enzymes, respectively. Incorporating sequences which arecleaved by proteolytic enzymes into chimeric proteins herein providescleavage at the linker site upon exposure to the appropriate enzyme andseparation of the two domains into separate entities. It is contemplatedthat numerous linker sequences can be incorporated into any of thechimeric proteins.

In another embodiment, a chimeric DNA construct includes a gene encodingan osteogenic protein or a fragment thereof linked to gene encoding anEMP or a fragment thereof. The gene sequence for various BMPs are known,see, e.g., U.S. Pat. Nos. 4,294,753, 4,761,471, 5,106,748, 5,187,076,5,141,905, 5,108,922, 5,116,738 and 5,168,050, each incorporated hereinby reference. A BMP-2B gene for use herein is synthesized by ligatingoligonucleotides encoding a BMP protein. The oligonucleotides encodingBMP-2B are synthesized using an automated DNA synthesizer (BeckmenOligo-1000). In preferred embodiment, the nucleotide sequence encodingthe BMP is maximized for expression in E. coli. This is accomplished byusing E. coli utilization tables to translate the sequence of aminoacids of the BMP into codons that are utilized most often by E. coli.Alternatively, native DNA encoding BMP isolated from mammals including-humans may be purified and used.

The BMP gene and the DNA sequence encoding an extracellular matrixprotein are cloned by standard genetic engineering methods as describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor 1989, hereby incorporated by reference.

The DNA sequence corresponding to the helical and telepeptide region ofcollagen I(α1) is cloned from a human fibroblast cell line. Two sets ofpolymerase chain reactions are carried out using cDNA prepared bystandard methods from AGQ2261A cells. The first pair of PCR primersinclude a 5′ primer bearing an XrnnI linker sequence and a 3′ primerbearing the BsmI site at nucleotide number 1722. The resulting PCRproduct consists of sequence from position 1 to 1722. The second pair ofprimers includes the BsmI site at 1722 and a linker sequence at the 3′end bearing a Bgl II site. The resulting PCR product consists ofsequence from position 1722 to 3196. The complete sequence is assembledby standard cloning techniques. The two PCR products are ligatedtogether at the Bsml site, and the combined clone is inserted into anyvector with XmnI-BglII sites such as pMAL-c2 vector.

To clone the BMP-2B gene, total cellular RNA is isolated from humanosteosarcoma cells (U-20S) by the method described by Robert E. FarrelJr. (Academic Press, CA, 1993 pp. 68-69) (herein incorporated byreference). The integrity of the RNA is verified by spectrophotometricanalysis and electrophoresis through agarose gels. Typical yields oftotal RNA are 50 μg from a 100 mm confluent tissue culture dish. The RNAis used to generate cDNA by reverse transcription using the Superscriptpre-amplification system by Gibco BRL. The cDNA is used as template forPCR amplification using upstream and downstream primers specific forBMP-2B (GenBank HUMBMP2B accession #M22490). The resulting PCR productconsists of BMP-2B sequence from position 1289-1619. The PCR product isresolved by electrophoresis through agarose gels, purified with geneclean (BIO 101) and ligated into pMal-c2 vector (New England Biolabs).The domain of human collagen I(α1) chain is cloned in a similar manner.However, the total cellular RNA is isolated from a human fibroblast cellline (AG02261A human skin fibroblasts).

A chimeric BMP/EMP DNA construct is obtained by ligating a synthetic BMPgene to a DNA sequence encoding an EMP such as collagen, fibrinogen,fibrin, fibronectin, elastin or laminin. However, chimeric polypeptidesherein are not limited to these particular proteins. FIGS. 14A-14C (SEQ.ID. NO. 7) illustrate a DNA construct which encodes a BMP-2B/collagenI(α1) chimeric protein. The coding sequence for an EMP may be ligatedupstream and/or downstream and in-frame with a coding sequence for theBMP. The DNA encoding an EMP may be a portion of the gene or an entireEMP gene. Furthermore, two different EMPs may be ligated upstream anddownstream from the BMP.

The BMP-2B/collagen I(α1) chimeric protein illustrated in FIGS. 14A-14Cincludes an XmnI linker sequence at base pairs (bp) 1-19, a collagendomain (bp 20-3190), a BglIII/BamHI linker sequence (bp 3191-3196), amature form of BMP2b (bp 3197-3529) and a HindII linker-sequence (bp3530-3535).

Any combination of growth factor and matrix protein sequences arecontemplated including repeating units, or multiple arrays of eachsegment in any order.

Incorporation of fragments of both matrix and growth factor proteins isalso contemplated. For example, in the case of collagen, only thehelical domain may be included. Other matrix proteins have defmeddomains, such as laminin, which has EGF-like domains. In these cases,specific functionalities can be chosen to achieve desired effects.Moreover, it may be useful to combine domains from disparate matrixproteins, such as the helical region of collagen and the cell attachmentregions of fibronectin. In the case of growth factors, specific segmentshave been shown to be removed from the mature protein by posttranslational processing. Chimeric proteins can be designed to includeonly the mature biologically active region. For example, in the case ofBMP-2B only the final 110 amino acids are found in the active protein.

In another embodiment, a transforming growth factor (TGF) moiety iscovalently linked with an EMP to form a chimeric protein. The TGF moietyincreases efficacy of the body's normal soft tissue repair response andalso induces osteogenesis. Consequently, TGF/EMP chimeric proteins maybe used for either or both functions. One of the fundamental propertiesof the TGF-βs is their ability to turn on various activities that resultin the synthesis of new connective tissue. See, Piez and Sporn eds.,Transforming Growth Factor-βs Chemistry, Biology and Therapeutics,Annals of the New York Academy of Sciences, Vol. 593, (1990). TGF-β isknown to exist in at least five different isoforms. The DNA sequence forHuman TGF-β₁ is known and has been cloned. See Derynck et al., HumanTransforming Growth Factor-Beta cDNA Sequence and Expression in TumourCell Lines, Nature, Vol. 316, pp. 701-705 (1985), herein incorporated byreference. TGF-β₂ has been isolated from bovine bone, human glioblastomacells and porcine platelets. TGF-B₃ has also been cloned. See ten Dijke,et al., Identification of a New Member of the Transforming GrowthFactor-β Gene Family, Proc. Natl. Acad. Sci. (USA), Vol. 85, pp.4715-4719 (1988) herein incorporated by reference.

A TGF-β/EMP chimeric protein incorporates the known activities of TGF-βsand provides integral scaffolding or substratum of the EMP as describedabove to yield a composition which further provides sustained releasefocal delivery at target sites.

The TGF-β moiety and the EMP moiety are optionally linked together bylinker sequences of amino acids. Linker sequences may be chosen basedupon particular properties which they impart to the chimeric protein.For example, amino acid sequences such as Ile-Glu-Glyn-Arg andLeu-Val-Pro-Arg are cleaved by Factor XA and Thrombin enzymes,respectively. Incorporating sequences which are cleaved by proteolyticenzymes into the chimeric protein provides cleavage at the linker siteupon exposure to the appropriate enzyme and separation of the domainsinto separate entities. FIG. 15 depicts an amino acid sequence for aTGF-β₁/collagen IA chimeric protein (SEQ. ID. NO. 8). The illustratedamino acid sequence includes the collagen domain (1-1057) and a matureform of TGF-β₁ (1060-1171).

A chimeric DNA construct includes a gene encoding TGF-β₁ or a fragmentthereof, or a gene encoding TGF-β₂ or a fragment thereof, or a geneencoding TGF-β₃ or a fragment thereof, ligated to a DNA sequenceencoding an EMP protein such as collagen (I-IV), fibrin, fibrinogen,fibronectin, elastin or laminin. A preferred chimeric DNA constructcombines DNA encoding TGF-β₁, a DNA linker sequence, and DNA encodingcollagen IA. A chimeric DNA construct containing TGF-β₁ gene and acollagen I(α1) gene is shown in FIGS. 16A-16C (SEQ. ID. NO. 9). Theillustrated construct includes an XmnI linker sequence (bp 1-19), DNAencoding a collagen domain (bp 20-3190), a BglII linker sequence (bp3191-3196), DNA encoding a mature form of TGF-β₁ (3197-3535), and anXbaI linker sequence (bp 3536-3541).

The coding sequence for EMP may be ligated upstream and/or downstreamand in-frame with a coding sequence for the TGFβ. The DNA encoding theextracellular matrix protein may encode a portion of a fragment of theEMP or may encode the entire EMP. Likewise, the DNA encoding the TGF-βmay be one or more fragments thereof or the entire gene. Furthermore,two or more different TGF-βs or two or more different EMPs may beligated upstream or downstream of alternate moieties.

In yet another embodiment, a dermatan sulfate proteoglycan mnoiety, alsoknown as decorin or proteoglycan II, is covalently linked with an EMP toform a chimeric protein. Decorin is known to bind to type I collagen andthus affect fibril formation, and to inhibit the cellattachment-promoting activity of collagen and fibrinogen by binding tosuch molecules near their cell binding sites. Chimeric proteins whichcontain a decorin moiety act to reduce scarring of healing tissue. Theprimary structure of the core protein of decorin has been deduced fromcloned cDNA. See Krusius et al., Primary Structure of an ExtracellularMatrix Proteoglycan Core Protein-Deduced from Cloned cDNA, Proc. Natl.Acad. Sci. (USA), Vol. 83, pp. 7683-7687 (1986) incorporated herein byreference.

A decorin/EMP chimeric protein incorporates the known activities ofdecorin and provides integral scaffolding or substratum of the EMP asdescribed above to yield a composition which allows sustained releasefocal delivery to target sites. FIGS. 17A-17B illustrate adecorin/collagen IA chimeric protein (SEQ.ID.NO.10) in which thecollagen domain includes amino acids 1-1057 and the decorin matureprotein incudes amino acids 1060-1388. FIG. 18 illustrates a decorinpeptide/collagen IA chimeric protein (SEQ. ID. NO. 11) in which thecollagen helical domain includes amino acids 1-1057 and the decorinpeptide fragment includes amino acids 1060-1107. The decorin peptidefragment is composed of P46 to G93 of the mature form of decorin.

Further provided is a chimeric DNA construct which includes a geneencoding decorin or one or more fragments thereof, optionally ligatedvia a DNA linker sequence to a DNA sequence encoding an EMP such ascollagen (I-IV), fibrin, fibrinogen, fibronectin, elastin or laminin. Apreferred chimeric DNA construct combines DNA encoding decorin, a DNAlinker sequence, and DNA encoding collagen I(α1). A chimeric DNAconstruct containing a decorin gene and a collagen I(α1) gene is shownin FIGS. 19A-19D (SEQ. ID. NO. 12). The illustrated construct includesan XmnI linker sequence (bp 1-19), DNA encoding a collagen domain (bp20-3190), a BglII linker sequence (bp 3191-3196), DNA encoding a matureform of decorin (bp 3197-4186) and a PstI linker sequence. A chimericDNA construct containing a decorin peptide gene and a collagen I(α1)gene is shown in FIGS. 20A-20C (SEQ. ID. NO. 13). The illustratedconstruct includes an XmnI linker sequence (bp 1-19), DNA encoding acollagen domain (bp 20-3190), a BglII linker sequence (bp 3191-3196),DNA encoding a peptide fragment of decorin (bp 3197-3343), and a PstIlinker sequence (bp 3344-3349).

The coding sequence for an EMP may be ligated upstream and/or downstreamand in-frame with a coding sequence for decorin. The DNA encoding theEMP may encode a portion or fragment of the EMP or may encode the entireEMP. Likewise, the DNA encoding decorin may be a fragment thereof or theentire gene. Furthermore, two or more different EMPs may be ligatedupstream and/or downstream from the DNA encoding decorin moiety.

Any of the above described chimeric DNA constructs may be incorporatedinto a suitable cloning vector. FIG. 21 depicts a pMal cloning vectorcontaining a polylinker cloning site. Examples of cloning vectors arethe plasmids pMal-p2 and-pMal-c2 (commercially available from NewEngland Biolabs). The desired chimeric DNA construct is incorporatedinto a polylinker sequence of the plasmid which contains certain usefulrestriction endonuclease sites which are depicted in FIG. 22 (SEQ. ID.NO. 14). The pMal-p2 polylinker sequence has XmnI, EcoRI, BamHI,HindIII, XbaI, SalI and PstI restriction endonuclease sites which aredepicted in FIG. 22. The polylinker sequence is digested with anappropriate restriction endonuclease and the chimeric construct isincorporated into the cloning vector by ligating it to the DNA sequencesof the plasmid. The chimeric DNA construct may be joined to the plasmidby digesting the ends of the DNA construct and the plasmid with the samerestriction endonuclease to generate “sticky ends” having 5′ phosphateand 3′ hydroxyl groups which allow the DNA construct to anneal to thecloning vector. Gaps between the inserted DNA construct and the plasmidare then sealed with DNA ligase. Other techniques for incorporating theDNA construct into plasmid DNA include blunt end ligation, poly(dA.dT)tailing techniques, and the use of chemically synthesized linkers. Analternative method for introducing the chimeric DNA construct into acloning vector is to incorporate the DNA encoding the extracellularmatrix protein into a cloning vector already containing a gene encodinga therapeutically active moiety.

The cloning sites in the above-identified polylinker site allow the cDNAfor the collagen I(α1)/BMP-2B chimeric protein illustrated in FIGS.14A-14C (SEQ. ID. NO. 7) to be inserted between the XmnI and the HindIIIsites. The cDNA encoding the collagen I(α1)/TGF-β₁ protein illustratedin FIGS. 16A-16C (SEQ. ID. NO. 9) is inserted between the XmnI and theXbaI sites. The cDNA encoding the collagen I(α1)/decorin proteinillustrated in FIGS. 19A-19D (SEQ. ID. NO. 12) inserted between the XmnIand the PstI sites. The cDNA encoding the collagen I(α1)/decorin peptideillustrated in FIGS. 20A-20C (SEQ. ID. NO. 13) is inserted between theXmnI and PstI sites.

Plasrnids containing the chimneric DNA construct are identified bystandard techniques such as gel electrophoresis. Procedures andmaterials for preparation of recombinant vectors, transformation of hostcells with the vectors, and host cell expression of polypeptides aredescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,supra. Generally, prokaryotic or eukaryotic host cells may betransforned with the recombinant DNA plasmids. Transformed host cellsmay be located through phenotypic selection genes of the cloning vectorwhich provide resistance to a particular antibiotic when the host cellsare grown in a culture medium containing that antibiotic.

Transformed host cells are isolated and cultured to promote expressionof the chimeric protein. The chimeric protein may then be isolated fromthe culture medium and purified by various methods such as dialysis,density gradient centrifugation, liquid column chromatography,isoelectric precipitation, solvent fractionation, and electrophoresis.However, purification of the chimeric protein by affinity chromatographyis preferred whereby the chimeric protein is purified by ligating it toa binding protein and contacting it with a ligand or substrate to whichthe binding protein has a specific affinity.

In order to obtain more effective expression of mammalian or humaneukaryotic genes in bacteria (prokaryotes), the mammalian or human genemay be placed under the control of a bacterial promoter. A proteinfusion and purification system is employed to obtain the chimericprotein. Preferably, any of the above-described chimeric DNA constructsis cloned into a pMal vector at a site in the vector's polylinkersequence. As a result, the chimeric DNA construct is operably fused withthe malE gene of the pMal vector. The malE gene encodes maltose bindingprotein (MBP). FIG. 23 depicts a pMal cloning vector containing aBMP/collagen DNA construct. A spacer sequence coding for 10 asparagineresidues is located between the maIE sequence and the polylinkersequence. This spacer sequence insulates MBP from the protein ofinterest. FIGS. 24, 25 and 26 depict pMal cloning vectors containing DNAencoding collagen chimeras with TGF-β₁, decorin and a decorin peptide,respectively. The pMal vector containing any of the chimeric DNAconstructs fused to the malE gene is transformed into E. coli.

The E. coli is cultured in a medium which induces the bacteria toproduce the maltose-binding protein fused to the chimeric protein. Thistechnique utilizes the P_(tac) promoter of the pMal vector. The MBPcontains a 26 amino acid N-terminal signal sequence which directs theMBP-chimeric protein through the E. coli cytoplasmic membrane. Theprotein can then be purified from the periplasm. Alternatively, thepMal-c2 cloning vector can be used with this protein fusion andpurification system. The pMal-c2 vector contains an exact deletion ofthe malE signal sequence which results in cytoplasmic expression of thefusion protein. A crude cell extract containing the fusion protein isprepared and poured over a colurnn of amylose resin. Since MBP has anaffinity for the amylose it binds to the resin. Alternatively, thecolumn can include any substrate for which MBP has a specific affinity.Unwanted proteins present in the crude extract are washed through thecolumn. The MBP fused to the chimeric protein is eluted from the columnwith a neutral buffer containing maltose or other dilute solution of adesorbing agent for displacing the hybrid polypeptide. The purifiedMBP-chimeric protein is! cleaved with a protease such as factor Xaprotease to cleave the MBP from the chimeric protein. The pMal-p2plasmid has a sequence encoding the recognition site for protease factorXa which cleaves after the amino acid sequence Isoleucine-Glutamicacid-Glycine-Arginine of the polylinker sequence.

The chimeric protein is then separated from the cleaved MBP by passingthe mixture over an amylose column. An alternative method for separatingthe MBP from the chimeric protein is by ion exchange chromatography.This system yields up to 100 mg of MBP-chimeric protein per liter ofculture. See Riggs, P., in Ausebel, F. M., Kingston, R. E., Moore, D.D., Seidman, J. G., Smith, J. A., Struhl, K. (eds.) Current Protocols inMolecular Biology, Supplement 19 (16.6.1-16.6.10) (1990) GreenAssociates/Wiley Interscience, New York, New England Biolabs (cat #800-65S 9pMALc2) pMal protein fusion and purification system herebyincorporated herein by reference. (See also European Patent No. 286 239herein incorporated by reference which discloses a similar method forproduction and purification of a protein such as collagen.)

Other protein fusion and purification systems may be employed to producechimeric proteins. Prokaryotes such as E. coli are the preferred hostcells for expression of the chimeric protein. However, systems whichutilize eukaryote host cell lines are also acceptable such as yeast,human, mouse, rat, hamster, monkey, amphibian, insect, algae, and plantcell lines. For example, HeLa (human epithelial), 3T3 (mousefibroblast), CHO (Chinese hamster ovary), and SP 2 (mouse plasma cell)are acceptable cell lines. The particular host cells that are chosenshould be compatible with the particular cloning vector that is chosen.

Another acceptable protein expression system is the BaculovirusExpression System manufactured by Invitrogen of San Diego, Calif.Baculoviruses form prominent crystal occlusions within the nuclei ofcells they infect. Each crystal occlusion consists of numerous virusparticles enveloped in a protein called polyhedrin. In the baculovirusexpression system, the native gene encoding polyhedrin is substitutedwith a DNA construct encoding a protein or peptide having a desiredactivity. The virus then produces large amounts of protein encoded bythe foreign DNA construct. The preferred cloning vector for use withthis system is pBlueBac III (obtained from Invitrogen of San Diego,Calif.). The baculovirus system utilizes the Autograph californicamultiple nuclear polyhidrosis virus (ACMNPV) regulated polyhedriripromoter to drive expression of foreign genes. The chimeric gene, i.e.,the. DNA construct encoding the chimeric protein, is inserted into thepBlueBac III vector immediately downstream from the baculoviruspolyhedrin promoter.

The pBlueBac III transfer vector contains a B-galactosidase reportergene which allows for identification of recombinant virus. TheB-galactosidase gene is driven by the baculovirus ETL promoter (P_(ETL))which is positioned in opposite orientation to the polyhedrin promoter(P_(PH) and the multiple cloning site of the vector. Therefore,recombinant virus coexpresses B-galactosidase and the chimeric gene.

Spodoptera frugiperda (Sf9) insect cells are then cotransfected withwild type viral DNA and the pBlueBac III vector containing the chimericgene. Recombination sequences in the pBlueBac III vector direct thevector's integration into the genome of the wild type baculovirus.Homologous recombination occurs resulting in replacement of the nativepolyhedrin gene of the baculovirus with the DNA construct encoding thechimeric protein. Wild type baculovirus which do not contain foreign DNAexpress the polyhedrin protein in the nuclei of the infected insectcells. However, the recombinants do not produce polyhedrin protein anddo not produce viral occlusions. Instead, the recombinants produce thechimeric protein.

Alternative insect host cells for use with this expression system areSf21-cell line derived from Spodoptera frugiperda and High Five celllines derived from Trichoplusia ni.

Other acceptable cloning vectors include phages, cosmids or artificialchromosomes. For example, bacteriophage lambda is a useful cloningvector. This phage can accept pieces of foreign DNA up to about 20,000base pairs in length. The lambda phage genome is a linear doublestranded DNA molecule with single stranded complementary (cohesive) endswhich can hybridize-with each other when inside an infected host cell.The lambda DNA is cut with a restriction endonuclease and the foreignDNA, e.g. the DNA to be cloned, is ligated to the phage DNA fragments.The resulting recombinant molecule is then packaged into infective phageparticles. Host cells are infected with the phage particles containingthe recombinant DNA. The phage DNA replicates in the host cell toproduce many copies of the desired DNA sequence.

Cosmids are hybrid plasmidfbacteriophage vectors which can be used toclone DNA fragments of about 40,000 base pairs. Cosmids are plasmidswhich have one or more DNA sequences called “cos” sites derived frombacteriophage lambda for packaging lambda DNA into infective phageparticles. Two cosmids are ligated to the DNA to be cloned. Theresulting molecule is packaged into infective lambda phage particles andtransfected into bacteria host cells. When the cosmids are inside thehost cell they behave like plasmids and multiply under the control of aplasmid origin of replication. The origin of replication is a sequenceof DNA which allows a plasmid to multiply within a host cell.

Yeast artificial chromosome vectors are similar to plasmids but allowfor the incorporation of much larger DNA sequences of about 400,000 basepairs. The yeast artificial chromosomes contain sequences forreplication in yeast. The yeast artificial chromosome containing the DNAto be cloned is transformed into yeast cells where it replicates therebyproducing many copies of the desired DNA sequence. Where phage, cosmids,or yeast artificial chromosomes are employed as cloning vectors,expression of the chimeric protein may be obtained by culturing hostcells that have been transfected or transformed with the cloning vectorin a suitable culture medium.

Chimeric proteins disclosed herein are intended for use in treatingmammals or other animals. The therapeutically active moieties describedabove, e.g., osteogenic agents such as BMPs, TGFs, decorin, and/orfragments of each of them, are all to be considered as being or havingbeen derived from physiologically active agents for purposes of thisdescription. The chimeric proteins and DNA constructs which incorporatea domain derived from one or more cellular physiologically active agentscan be used for in vivo therapeutic treatment, in vitro research or fordiagnostic purposes in general.

When used in vivo, formulations containing the present chimeric proteinsmay be placed in direct contact with viable tissue, including bone, toinduce or enhance growth, repair and/or replacement of such tissue. Thismay be accomplished by applying a chimeric protein directly to a targetsite during surgery. It is contemplated that minimally invasivetechniques such as endoscopy are to be used to apply a chimeric proteinto a desired location. Formulations containing the chimeric proteinsdisclosed herein may consist solely of one or more chimeric proteins ormay also incorporate one or more pharmaceutically acceptable adjuvants.

In an alternate embodiment, any of the above-described chimeric proteinsmay be contacted with, adhered to, or otherwise incorporated into animplant such as a drug delivery device or a prosthetic device. Chimericproteins may be microencapsulated or macroencapsulated by liposomes orother membrane forming materials such as alginic acid derivatives priorto implantation and then implanted in the form of a pouchlike implant.The chimeric protein may be microencapsulated in structures in the formof spheres, aggregates of core material embedded in a continuum of wallmaterial or capillary designs. Microencapsulation techniques are wellknown in the art and are described in the Encyclopedia of PolymerScience and Engineering, Vol. 9, pp. 724 et seq. (1980) herebyincorporated herein by reference.

Chimeric proteins may also be coated on or incorporated into medicallyuseful materials such as meshes, pads, felts, dressings or prostheticdevices such as rods, pins, bone plates, artificial joints, artificiallimbs or bone augmentation implants. The implants may, in part, be madeof biocompatible materials such as glass, metal, ceramic, calciumphosphate or calcium carbonate based materials. Implants havingbiocompatible biomaterials are well known in the art and are allsuitable for use herein. Implant biomaterials derived from naturalsources such as protein fibers, polysaccharides, and treated naturallyderived tissues are described in the Encyclopedia of Polymer Science andEngineering, Vol. 2, pp. 267 et seq. (1989) hereby incorporated hereinby reference. Synthetic biocompatible polymers are well known in the artand are also suitable implant materials. Examples of suitable syntheticpolymers include urethanes, olefins, terephthalates, acrylates,polyesters and the like. Other acceptable implant materials arebiodegradable hydrogels or aggregations of closely packed particles suchas polymethylmethacrylate beads with a polymerized hydroxyethylmethacrylate coating. See the Encyclopedia of Polymer Science andEngineering, Vol. 2, pp. 267 et seq. (1989) hereby incorporated hereinby reference.

The chimeric protein herein provides a useful way for immobilizing orcoating a physiologically active agent on a pharmaceutically acceptablevehicle to deliver the physiologically active agent to desired sites inviable tissue. Suitable vehicles include those made of bioabsorbablepolymers, biocompatible nonabsorbable polymers, lactoner putty andplaster of Paris. Examples of suitable bioabsorbable and biocompatiblepolymers include homopolymers, copolymers and blends of hydroxyacidssuch as lactide - and glycolide, other absorbable polymers which may beused alone or in combination with hydroxyacids including dioxanones,carbonates such as trimethylene carbonate, lactones such ascaprolactone, polyoxyalkylenes, and oxylates. See the Encyclopedia ofPolymer Science and Engineering, Vol. 2, pp. 230 et seq. (1989) herebyincorporated herein by reference.

These vehicles may be in the form of beads, particles, putty, coatingsor film vehicles. Diffusional systems in which a core of chimericprotein is surrounded by a porous membrane layer are other acceptablevehicles.

In another aspect, the amount of amino acid analog(s) transport into atarget cell can be regulated by controlling the tonicity of the growthmedia. A hypertonic growth media increases uptake oftrans4-hydroxyproline into E. coli as illustrated in FIG. 2A. All knownmethods of increasing osmolality of growth media are appropriate for useherein including addition of salts such as sodium chloride, KCI, MgCl₂and the like, and sugars such as sucrose, glucose, maltose, etc. andpolymers such as polyethylene glycol (PEG), dextran, cellulose, etc. andamino acids such as glycine. Increasing the osmolality of growth mediaresults in greater intracellular concentration of amino acid analog(s)and a higher degree of complexation of amino acid analog(s) to tRNA. Asa consequence, proteins produced by the cell achieve a higher degree ofincorporation of amino acid analogs. FIG. 12 illustrates percentage ofincorporation of proline and hydroxyproline into MBP under isotonic andhypertonic media conditions in comparison to proline in native MBP.Thus, manipulating osmolality, in addition to adjusting concentration ofamino acid analog(s) in growth media allows a dual-faceted approach toregulating their uptake into prokaryotic cells and eukaryotic cells asdescribed above and consequent incorporation into target polypeptides.

Any growth media can be used herein including commercially availablegrowth media such as M9 minimal medium (available from Gibco LifeTechnologies, Inc.), LB medium, NZCYM medium, terrific broth, SOB mediumand others that are well known in the art.

Collagen from different tissues can contain different amounts oftrans-4-hydroxyproline. For example, tissues that require greaterstrength such as bone contain a higher number of trans-4-hydroxyprolineresidues than collagen in tissues requiring less strength, e.g., skin.The present system provides a method of adjusting the amount oftrans-4-hydroxyproline in collagen, collagen fragments, collagen-likepeptides, and chimeric peptides having a collagen domain, collagenfragment domain or collagen-like peptide domain fused to aphysiologically active domain, since by increasing or decreasing theconcentration of trans-4-hydroxyproline in growth media, the amount oftrans-4-hydroxyproline incorporated into such polypeptides is increasedor decreased accordingly. The collagen, collagen fragments,collagen-like peptides and above-chimeric peptides can be expressed withpredetermined levels of trans-4-hydroxyproline. In this manner physicalcharacteristics of an extracellular matrix can be adjusted based uponrequirements of end use. Without wishing to be bound by any particulartheory, it is believed that incorporation of trans-4-hydroxyproline intothe EMP moieties herein provides a basis for self aggregation asdescribed herein.

In another aspect, the combination of incorporation oftrans-4-hydroxyproline into collagen and fragments thereof usinghyperosmotic media and genes which have been altered such that codonusage more closely reflects that found in E. coli, but retaining theamino acid sequence found in native human collagen, surprisinglyresulted in production by E. coli of human collagen and fragmentsthereof which were capable of self aggregation.

The human collagen Type I (α₁) gene sequence (FIG. 27A-27E) (SEQ. ID.NO. 15) contains a large number of glycine and proline codons (347glycine and 240 proline codons) arranged in a highly repetitive manner.Table I below is a codon frequency tabulation for the human Type I (α₁)collagen gene. Of particular note is that the GGA glycine codon occurs64 times and the CCC codon for proline occurs 93 times. Both of thesecodons are considered to be rare codons in E. coli. See, Sharp, P. M.and W.-H. Li. Nucleic Acids Res. 14: 7737-7749, 1986. These, and similarconsiderations for other human collagen genes are shown herein toaccount for the difficulty in expressing human collagen genes in E.coli. TABLE 1 Codon Count % age Codon Count % age TTT-Phe 1 0.09 TCT-Ser18 1.70 TTC-Phe 14 1.32 TCC-Ser 4 0.37 TTA-Leu 0 0.00 TCA-Ser 2 0.18TTG-Leu 3 0.28 TCG-Ser 0 0.00 CTT-Leu 4 0.37 CCT-Pro 141 13.33 CTC-Leu 70.66 CCC-Pro 93 8.79 CTA-Leu 0 0.00 CCA-Pro 6 0.56 CTG-Leu 7 0.66CCG-Pro 0 0.00 ATT-Ile 6 0.56 ACT-Thr 11 1.04 ATC-Ile 0 0.00 ACC-Thr 40.37 ATA-Ile 1 0.09 ACA-Thr 2 0.18 ATG-Met 7 0.66 ACG-Thr 0 0.00 GTT-Val10 0.94 GCT-Ala 93 8.79 GTC-Val 5 0.47 GCC-Ala 24 2.27 GTA-Val 0 0.00GCA-Ala 6 0.56 GTG-Val 5 0.47 GCG-Ala 0 0.00 TAT-Tyr 2 0.18 TGT-Cys 00.00 TAC-Tyr 2 0.18 TGC-Cys 0 0.00 TAA-*** 0 0.00 TGA-*** 0 0.00 TAG-***0 0.00 TGG-Trp 0 0.00 CAT-His 0 0.00 CGT-Arg 26 2.45 CAC-His 3 0.28CGC-Arg 6 0.56 CAA-Gln 13 1.22 CGA-Arg 11 1.04 CAG-Gln 17 1.60 CGG-Arg 10.09 AAT-Asn 6 0.56 AGT-Ser 4 0.37 AAC-Asn 5 0.47 AGC-Ser 11 1.04AAA-Lys 19 1.79 AGA-Arg 9 0.85 AAG-Lys 19 1.79 AGG-Arg 0 0.00 GAT-Asp 232.17 GGT-Gly 174 16.46 GAC-Asp 11 1.04 GGC-Gly 97 9.17 GAA-Glu 24 2.27GGA-Gly 64 6.05 GAG-Glu 25 2.36 GGG-Gly 11 1.04

In a first step, the sequence of the heterologous collagen gene ischanged to reflect the codon bias in E. coli as given in codon usagetables (e.g. Ausubel et al., (1995) Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y.; Wada et al., 1992, supra).Rare E. coli codons (See, Sharp, P. M. and W.-H. Li; Nucleic Acids Res.14: 7737-7749, 1986) are avoided. Second, unique restriction enzymesites are chosen that are located approximately every 120-150 base pairsin the sequence. In certain cases this entails altering the nucleotidesequence but does not change the amino acid sequence. Third, oligos ofapproximately 80 nucleotides are synthesized such that when two sucholigos are annealed together and extended with a DNA polymerase theyreconstruct a approximately 120-150 base pair section of the gene (FIG.28). The section of the gene encoding the very amino terminal portion ofthe protein has an initiating methionine (ATG) codon at the 5′ end and aunique restriction site followed by a stop (TAAT) signal at the 3′ end.The remaining sections have unique restriction sites at the 5′ end andunique restriction sites followed by a TAAT stop signal the 3′ end. Thegene is assembled by sequential addition of each section to thepreceding 5′ section. In this manner, each successively larger sectioncan be independently constructed and expressed. FIG. 28 is a schematicrepresentation of the construction of the human collagen gene startingfrom synthetic oligos.

A fragment of the human Type I α1 collagen chain fused to the C-terminusof glutathione S-transferase (GST-D4, FIG. 29) (SEQ. ID. NO. 18) wasprepared and tested for expression in E. coli strain JM109 (F⁻) underconditions of hyperosmotic shock. The collagen fragment included theC-terminal 193 amino acids of the triple helical region and the 26 aminoacid C-terminal telopeptide. FIG. 29 is a schematic of the amino acidsequence of the GST-ColECol (SEQ. ID. NO. 17) and GST-D4 (SEQ. ID. NO.18) fusion proteins. ColECol comprises the 17 amino acid N-terminaltelopeptide, 338 Gly-X-Y repeating tripeptides, and the 26 amino acidC-terminal telopeptide. There is a unique methionine at the junction ofGST and D4, followed by 64 Gly-X-Y repeats, and the 26 amino acidtelopeptide. The residue (Phel99) in the C-terminal telopeptide of D4where pepsin cleaves is indicated. The gene was synthesized for thecollagen fragment from synthetic oligonucleotides designed to reflectoptimal E. coli usage. FIG. 30 is a table depicting occurrence of thefour proline and four glycine codons in the human Type I al gene (HCol)and the Type I a I gene with optimized E. coli codon usage (ColECol).Usage of the remaining codons in ColECol was also optimized for E. coliexpression according to Wada et al., supra. Protein GST-D4 wasefficiently expressed in JM109 (F⁻) in minimal media lacking proline butsupplemented with Hyp and NaCl (See FIGS. 31 and 32). Expression wasdependent on induction with isopropyl-1-thio-β-galactopyranoside (IPTG),trans-4-hydroxyproline and NaCl. At a fixed NaCl concentration of 500mM, expression was minimal at trans-4-hydroxyproline concentrationsbelow −20 mM while the expression level plateaued attrans-4-hydroxyproline concentrations above 40 mM. See FIG. 31 whichdepicts a gel showing expression and dependence of expression of GST-D4on hydroxyproline. The concentration of hydroxyproline is indicatedabove each lane. Osmolyte (NaCl) was added at 500 mM in each culture andeach was induced with 1.5 mM IPTG. The arrow marks the position ofGST-D4. Likewise, at a fixed trans-4-hydroxyproline concentration of 40mM, NaCl concentrations below 300 mM resulted in little proteinaccumulation and expression decreased above 700-800 mM NaCl. See FIG. 32which depicts a gel showing expression of GST-D4 in hyperosmotic media.Lanes 2 and 3 are uninduced and induced samples, respectively, eachwithout added osmolyte. The identity and quantity of osmolyte isindicated above each of the other lanes. Trans-4-Hydroxyproline wasadded at 40 mM in each culture and all cultures except that in lane 1were induced with 1.5 mM IPTG. The arrow marks the position of GST-D4.

Either sucrose or KCl can be substituted for NaCl as the osmolyte (SeeFIG. 32). Thus, the osmotic shock-mediated intracellular accumulation oftrans-4-hydroxyproline was a critical determinant of expression ratherthan the precise chemical identity of the osmolyte. Despite the largenumber of prolines (66) in GST-D4, its size (46 kDA), and non-optimalgrowth conditions, it was expressed at ˜10% of the total cellularprotein. Expressed proteins of less than full-length indicative ofaborted transcription, translation, or mRNA instability were notdetected.

The gene for protein D4 contains 52 proline codons. In the expressionexperiments reflected in FIGS. 31 and 32, it was expected thattrans-4-hydroxyproline would be inserted at each of these codonsresulting in a protein where trans-4-hydroxyproline had been substitutedfor all prolines. To confirm this, GST-D4 was cleaved with BrCN in 0.1 NHCl at methionines within GST and at the unique methionine at theN-terminal end of D4, and D4 purified by reverse phase HPLC. CrudeGST-D4 was dissolved in 0.1 M HCl in a round bottom flask with stirring.Following addition of a 2-10 fold molar excess of clear, crystallineBrCN, the flask was evacuated and filled with nitrogen. Cleavage wasallowed to proceed for 24 hours, at which time the solvent was removedin vacuo. The residue was dissolved in 0.1% trifluoroacetic acid (TFA)and purified by reverse-phase HPLC using a Vydac C4 RP-HPLC column(10×250 mm, 5 μ, 300 Å) on a BioCad Sprint system (PerceptiveBiosystems, Framingham, Mass.). D4 was eluted with a gradient of 15 to40% acetonitrile/0.1% TFA over a 45 min. period. D4 eluted as a singlepeak at 26% acetonitrile/0.1% TFA. Standard BrCN cleavage conditions(70% formic acid) resulted in extensive formylation of D4, presumably atthe hydroxyl groups of the trans-4-hydroxyproline residues. Formylationof BrCN/formic acid-cleaved proteins had been noted before (Beavis etal., Anal. Chem., 62, 1836 (1990)). Amino acid analysis was carried outon a Beckman ion exchange instrument with post-column derivatization.N-terminal sequencing was performed on an Applied Biosystems sequencerequipped with an on-line HLPC system. Electrospray mass spectra wereobtained with a VG Biotech BIO-Q quadropole analyzer by M-Scan, Inc.(West Chester, Pa.). For CD thermal melts, the temperature was raised in0.5° C. increments from 4° C. to 85° C. with a four minute equilibrationbetween steps. Data were recorded at 221.5 nm. The thermal transitionwas calculated using the program ThermoDyne (MORE). The electrospraymass spectroscopy of this protein gave a single molecular ioncorresponding to a mass of 20,807 Da. This mass is within 0.05% of thatexpected for D4 if it contains 100% trans-4-hydroxyproline in lieu ofproline. Proline was not detected in amino acid analysis of purified D4,again consistent with complete substitution of trans-4-hydroxyprolinefor proline. To confirm further that trans-4-hydroxyproline substitutionhad only occurred at proline codons, the N-terminal 13 amino acids of D4was sequenced as above. The first 13 codons of D4 specify the proteinsequence H₂N-Gly-Pro-Pro-Gly-Leu-Ala-Gly-Pro-Pro-Gly-Glu-Ser-Gly (SEQ.ID. NO. 41). The sequence found wasH₂N-Gly-Hyp-Hyp-Gly-Leu-Ala-Gly-Hyp-Hyp-Gly-Glu-Ser-Gly (SEQ. ID. NO.42), see FIG. 69. Taken together, these results indicate thattrans-4-hydroxyproline (Hyp) was inserted only at proline codons andthat the fidelity of the E. coli translational machinery was nototherwise altered by either the high intracellular concentration ortrans-4-hydroxyproline or hyperosmotic culture conditions.

To determine whether D4, containing trans-4-hydroxyproline in both the Xand Y positions, forms homotrimeric helices and to compare stability tonative collagen, the following was noted: In neutral pH phosphatebuffer, D4 exhibits a circular dichroism (CD) spectrum characteristic ofa triple helix (See FIG. 33 and Bhatnagar et al., Circular Dichroism andthe Conformational Analysis of Biomolecules, G. D. Fasman, Ed. PlenumPress, New York, (1996 p. 183). FIG. 33 illustrates circular dichroismspectra of native and heat-denatured D4 in neutral phosphate buffer.HPLC-purified D4 was dissolved in 0.1M sodium phosphate, pH 7.0, to afinal concentration of 1 mg/mL (E²⁸⁰=3628 M⁻¹cm⁻¹). The solution wasincubated at 4° C. for two days to allow triple helices to form prior toanalysis. Spectra were obtained on an Aviv model 62DS spectropolarimeter(Yale University, Molecular Biophysics and Biochemistry Department). A 1mm path length quartz suprasil fluorimeter cell was used. Following a 10min incubation period at 4° C., standard wavelength spectra wererecorded from 260 to 190 nm using 10 sec acquisition times and 0.5 nmscan steps. This spectrum is characterized by a negative ellipticity at198 nm and a positive ellipticity at 221 nm. The magnitudes of both ofthese absorbances was greater in neutral pH buffer compared to acidicconditions. Comparable dependence of stability on pH has been noted forcollagen-like triple helices. See, e.g., Venugopal et al., Biochemistry,33, 7948 (1994). Heating at 85° C. for five minutes prior to obtainingthe CD spectrum decreased the magnitude of the absorbance at 198 nm andabolished the absorbance at 221 nm (FIG. 33). This behavior is alsotypical of the triple helical structure of collagen. See, R. S.Bhatnagar et al., Circular Dichroism and the Conformational Analysis ofBiomolecules G. D. Fasffan, Ed., supra. A thermal melt profile of D4conducted as above in phosphate buffer gave a melting temperature ofabout 29° C. A fragment of the C-terminal region of the bovine Type I alcollagen chain comparable in length to D4 forms homotrimeric heliceswith a melting temperature of 26° C. (See, A. Rossi, et al.,Biochemistry 35, 6048 (1996)).

Resistance to pepsin digestion is a second commonly used indication oftriple helical structure. At 4° C., the majority of D4 is digestedrapidly by pepsin to a protein of slightly lower molecular weight. FIG.34 is a gel illustrating the result of digestion of D4 with bovinepepsin. Purified D4 was dissolved in 0.1 M sodium phosphate, pH 7.0, to1.6 μg/μl and incubated at 4° C. for 7 days. Aliquots (10 μl) wereplaced into 1.5 ml centrifuge tubes and adjusted with water and 1 Macetic acid solutions to 25 μl final volume and 200 mM final acetic acidconcentration. Each tube was then incubated for 20 min. at the indicatedtemperature and pepsin (0.5 μl of a 0.25 μg/μl solution) was added toeach tube and digestion allowed to proceed for 45 minutes. Followingdigestion, samples were quenched with loading buffer and analyzed bySDS-PAGE. However, the initial pepsin cleavage product is resistant tofurther digestion up to −30° C. Amino terminal sequencing as above ofthe initial pepsin cleavage product showed that the N-terminus wasidentical to that of full-length D4. Mass spectral analysis as above ofthe digestion product gave a parent ion with a molecular weightconsistent with cleavage in the C-terminal telopeptide on the N-terminalside of Phel19 (See FIG. 29) suggesting that this portion of the proteinis either globular or of ill-defined structure and rapidly cleaved bypepsin while the triple helical region is resistant to digestion. Thus,despite global trans-4-hydroxyproline for proline substitution in boththe X and Y positions, D4 formed triple helices of stability similar tocomparably sized fragments of bovine collagen containing Hyp at thenormal percentage and only in the Y position.

The full-length human Type I al collagen chain, although more than fourtimes the size of D4, also expressed as a N-terminal fusion with GST(GST-ColECol, FIG. 29) in JM109(F⁻) in Hyp/NaCl media. FIG. 35 is a geldepicting expression of GST-HCol and GST-ColECol. Trans-4-hydroxyprolinewas added at 40 mM and NaCl at 500 mM. Expression was induced with 1.5mM IPTG. The arrow marks the position of GST-ColECol. In the proceduresresulting in the gels shown in FIGS. 31, 32 and 35, five ml cultures ofJM109 (F⁻) harboring the expression plasmid in LB media containing 100μg/ml ampicillin were grown overnight. Cultures were centrifuged and thecell pellets washed twice with five ml of M9/Amp media (See, J.Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989))supplemented with 0.5% glucose and 100 μg/ml of all amino acids exceptglycine and alanine which were at 200 μg/ml and containing no proline.The cells were finally resuspended in five ml of the above media.Following incubation at 37° C. for 30 min., hydroxyproline, osmolyte, orIPTG were added as indicated. After four hours, aliquots of the cultureswere analyzed by SDS-PAGE.

Like D4, the gene for protein ColECol was constructed from syntheticoligonucleotides designed to mimic codon usage in highly-expressed E.coli genes. In contrast to GST-ColECol, expression from a GST-human TypeI α1 gene fusion (pHCol) identical to GST-ColECol in coded amino acidsequence but containing the human codon distribution could not bedetected in Coomassie blue-stained SDS-PAGE gels of total cell lysatesof induced JM109 (F⁻)/pHCol cultures (FIG. 35). The gene for the Type Iα1 collagen polypeptide was cloned by polymerase chain reaction of thegene from mRNA isolated from human foreskin cells (HS27, ATCC 1634) withprimers designed from the published gene sequence (GenBank Z74615). The5′ primer added a flanking EcoR I recognition site and the 3′ primer aflanking Hind III recognition site. The gene was cloned into the EcoRI/Hind III site of plasmid pBSKS⁺ (Stratagene, La Jolla, Calif.), fourmutations corrected using the ExSite mutagenesis kit (Stratagene, LaJolla, Calif.), the sequence confirmed by dideoxy sequencing, andfinally the EcoR I/Xho I fragment subcloned into plasmid pGEX4T.1(Pharmacia, Piscataway, N.J.). The GST-HCol gene is expression-competentbecause a protein of the same molecular weight as GST-ColECol isdetected when immunoblots of total cell lysates are probed with ananti-Type I collagen antibody. Thus, sequence or structural differencesbetween the genes for ColECol and HCol are critical determinants ofexpression efficiency in E. coli. This is likely due to the codondistribution in these genes and ultimately to differences in tRNAisoacceptor levels in E. coli compared to humans. GST-ColECol, GST-D4,and GST-HCol do not accumulate in hyperosmotic shock media when prolineis substituted for hydroxyproline or in rich media. A possibleexplanation is that the trans-4-hydroxyproline-containing proteins maybe resistant to degradation because they fold into a protease-resistanttriple helix while the proline-containing proteins do not adopt thisstructure. The large number of codons non-optimal for E. coli found inthe human gene and the instability of proline-containing collagen in E.coli may, in part, explain why expression of human collagen in E. colihas not been previously reported.

As discussed above, collagen mimetic polypeptides, i.e., engineeredpolypeptides having certain compositional and structural traits incommon with collagen are also provided herein. Such collagen mimeticpolypeptides may also be made to incorporate amino acid analogs asdescribed above. GST-CM4 consists of glutathione S-transferase fused to30 repeats of a Gly-X-Y sequence. The Gly-X-Y repeating section mimicsthe Gly-X-Y repeating unit of human collagen and is referred to ascollagen mimetic 4 or CM4 herein. Thus, the hydroxyproline-incorporatingtechnology was also demonstrated to work with a protein and DNA sequenceanalogous to that found in human collagen. Amino acid analysis ofpurified CM4 protein express in E. coli strain JM109 (F—) underhydroxyproline-incorporating conditions compared to analysis of the sameprotein expressed under proline-incorporating conditions, demonstratesthat the techniques herein result in essentially complete substitutionof hydroxyproline for proline. The amino acid analysis was performed onCM4 protein that had been cleaved from and purified away from GST. Thisremoves any possible ambiguities associated with the fusion protein.

Expression in media containing at least about 200 mM NaCl is preferableto accumulate significant amount of protein containing hydroxyproline. Aconcentration of about 400-500 mM NaCl appears to be optimal. EitherKCl, sucrose or combinations thereof may be used in substitution of orwith NaCl. However, expression in media without an added osmolyte (i.e.under conditions that more closely mimic those of Deming et al., In VivoIncorporation of Proline Analogs into Artificial Protein, Poly. Mater.Sci. Engin. Proceed., supra) did not result in significant expression ofhydroxyproline-containing proteins in JMO109 (F—). This is illustratedin FIG. 36 which is a scan of a SDS-PAGE gel showing the expression ofGST-CM4 in media with or without 500 mM NaCl and containing eitherproline or hydroxyproline. The SDS-PAGE gel reflects 5 hourpost-induction samples of GST-CM4 expressed in JM109 (F—). Equivalentamounts, based on OD600nm, of each culture were loaded in each lane.Gels were stained with Coomasie Blue, destained, and scanned on a PDI420oe scanner. Lane 1: 2.5mM proline/0 mM NaCl. Lane 2: 2.5 mMproline/500 mM NaCl. Lane 3: 80 mM hydroxyproline/0 mM NaCl. Lane 4: 80mM hydroxyproline/500 mM NaCl. Lane 5: Molecular weight markers. Thelower arrow indicates the migration position of proline-containingGST-CM4 in lanes 1 and 2. The upper arrow indicates the migrationposition of hydroxyproline-containing GST-CM4 in lanes 3 and 4. Notethat GST-CM4 expressed in the presence of hydroxyproline runs at ahigher apparent molecular weight (compare lanes 1 and 4). This isexpected since hydroxyproline is of greater molecular weight thanproline. If all the prolines in GST-CM4 are substituted withhydroxyproline, the increase in molecular weight is 671 Da (+2%). Notealso that protein expressed in the presence of proline accumulates incultures irrespective of the NaCl concentration (compare lanes 1 and 2).In contrast, significant expression in the presence of hydroxyprolineonly occurs in the culture containing 500 mM NaCl (compare lanes 3 and4). FIG. 37 further illustrates the dependence of expression on NaClconcentration by showing that significant expression of GST-CM4 occursonly at NaCl concentration greater than 200 mM. The SDS-PAGE gelreflects 6 hour post-induction samples of GST-CM4 expressed in JM109(F—) with varying concentrations of NaCl. All cultures contained 80 mMhydroxyproline. Lane 1: 500 mM NaCl, not induced. Lanes 2-6: 500 mM, 400mM, 300 mM, 200 mM, and 100 mM NaCl, respectively. All induced with 1.5mM IPTG. Lane 7: Molecular weight markers. The arrow indicates themigration position of hydroxyproline-containing GST-CM4. FIG. 38 is ascan of an SDS-PAGE gel of expression of GST-CM4 in either 400 mM NaClor 800 mM sucrose. The SDS-PAGE gel reflects 4 hour post-inductionsamples of GST-CM4 expressed in JM109 (F—). All cultures contained 80 mMhydroxyproline and all, except that electrophoresed in lane 2, contained400 mM NaCl. Lane 2 demonstrates expression in sucrose in lieu of NaCl.Lane 1: Molecular weight markers. Lane 2: 800 mM sucrose (no NaCl).Lanes 3-9: 0 mM, 0.025 mM, 0.1 mM, 0.4 mM, 0.8 mM, 1.25 mM, 2.5 mMproline, respectively. The upper arrow indicates the migration positionof hydroxyproline-containing GST-CM4 and the lower arrow indicates themigration position of proline-containing GST-CM4. Expression is apparentin both cases (compare lanes 2 and 3).

If expression of GST-CM4, as described in Example 17 below, is performedin varying ratios of hydroxyproline and proline the expressed proteinappears to contain varying amounts of hydroxyproline. Thus, if onlyhydroxyproline is present during expression, a single expressed proteinof the expected molecular weight is evident on a SDS-PAGE gel (FIG. 38,lane 3). If greater than approximately 1 mM proline is present, again asingle expressed protein is evident, but at a lower apparent molecularweight, as expected for the protein containing only proline (FIG. 38,lanes 7-9). If lesser amount of proline are used during expression,species of apparent molecular weight intermediate between these extremesare evident. This phenomenon, evident as a “smear” or “ladder” ofproteins running between the two molecular weight extremes on anSDS-PAGE gel, is illustrated in lanes 3-9 of FIG. 38. Lanes 3-9 on thisgel are proteins from expression in a fixed concentration of 80 mMhydroxyproline and 400 mM NaCl. However, in moving from lane 3 to 9 theproline concentration increases from none (lane 3) to 2.5 mM (lane 9)and expression shifts from a protein of higher molecular weight(hydroxyproline-containing GST-CM4) to lower molecular weight(proline-containing GST-CM4). At proline concentrations of 0.025 mM and0.1 mM, species of intermediate molecular weight are apparent (lanes 4and 5). This clearly demonstrates that the percent incorporation ofhydroxyproline in an expressed protein can be controlled by expressionin varying ratios of analogue to amino acid.

Proline starvation prior to hydroxyproline incorporation is an importanttechnique used herein. It insures that no residual proline is presentduring expression to compete with hydroxyproline. This enablesessentially 100% substitution with the analogue. As shown in FIG. 38,starvation conditions allow expression under precisely controlled ratiosof proline and hydroxyproline. The amount of hydroxyproline vs. prolineincorporated into the recombinant protein can therefore be controlled.Thus, particular properties of the recombinant protein that depend uponthe relative amount of analogue incorporated can be tailored by thepresent methodology to produce polypeptides with unique and beneficialproperties.

Human collagen, collagen fragments, collagen-like peptides (collagenmimetics) and the above chimeric polypeptides produced by recombinantprocesses have distinct advantages over collagen and its derivativesobtained from non-human animals. Since the human gene is used, thecollagen will not act as a xenograft in the context of a medicalimplant. Moreover, unlike naturally occurring collagen, the extent ofproline hydroxylation can be predetermined. This unprecedented degree ofcontrol permits detailed investigation of the contribution oftrans-4-hydroxyproline to triple helix stabilization, fibril formationand biological activity. In addition, design of medical implants basedupon the desired strength of collagen fibrils is enabled.

The following examples are included for purposes of illustration and arenot to be construed as limitations herein.

EXAMPLE 1 Trans-Membrane Transport

A 5 mL culture of E. coli strain DH5α (supE44 ΔlacU169 (φ801acZ ΔM15)hsdR17 recA1 endA1 gyrA96 thi-1 relA1) containing a plasmid conferringresistance to ampicillin (pMAL-c2, FIG. 1) was grown in Luria Broth toconfluency (˜16 hours from inoculation). These cells were used toinoculate a 1 L shaker flask containing 500 mL of M9 minimal medium (M9salts, 2% glucose, 0.01 mg/mL thiamine, 100 ∥g/mL ampicillinsupplemented with all amino acids at 20 ∥g/mL) which was grown to anAU₆₀₀ of 1.0 (18-20 hours). The culture was divided in half and thecells harvested by centrifugation. The cells from one culture,were-resuspended in 250 mL M9 media and those from the other in 250 mLof M9 media containing 0.5M NaCl. The cultures were equilibrated in anair shaker for 20 minutes at 37° C. (225 rpm) and divided into ten 25 mLaliquots. The cultures were returned to the shaker and 125 μl of 1Mhydroxyproline in distilled H₂O was added to each tube. At 2, 4, 8, 12,and 20 minutes, 4 culture tubes (2 isotonic, 2 hypertonic) were vacuumfiltered onto 1 μm polycarbonate filters that were immediately placedinto 2 mL microfuge tubes -containing 1.2 mL of 0.2M NaOH/2% SDS indistilled H₂O. After overnight lysis, the filters were carefully removedfrom the tubes, and the supernatant buffer was assayed forhydroxyproline according to the method of Grant, Journal of ClinicalPathology, 17:685 (1964). The intracellular concentration oftrans-4-hydroxyproline versus time is illustrated graphically in FIG. 2.

EXAMPLE 2 Effects of Salt Concentration on Transmembrane Transport

To determine the effects of salt concentration on transmembranetransport, an approach similar to Example 1 was taken. A S mL culture ofE. coli strain DH5α (supE44 ΔlacU169 (φ80lacZ ΔM15) hsdR17 recA1 entA1gyrA96 thi-1 relA1) containing a plasmid conferring resistance toampicillin (pMAL-c2, FIG. 1) was grown in Luria Broth to confluency (˜16hours from inoculation). These cells were used to inoculate a 1 L shakerflask containing 500 mL of M9 minimal medium (M9 salts, 2% glucose, 0.01mg/nl thiamine, 100 μg/mL ampicillin supplemented with all amino acidsat 20 μg/mL) that was then grown to an AU₆₀₀ of 0.6. The culture wasdivided into three equal parts, the cells in each collected bycentrifugation and resuspended in 150 mL M9 media, 150 mL M9 mediacontaining 0.5M NaCl, and 150 mL M9 media containing 1.0M NaCl,respectively. The cultures were equilibrated for 20 minutes on a shakerat 37° C. (225 rpm) and then divided into six 25 mL aliquots. Thecultures were returned to the shaker and 125 μL of 1M hydroxyproline indistilled H₂O was added to each tube. At 5 and 15 minutes, 9 culturetubes (3 isotonic, 3×0.5M NaCl, and 3×1.0M NaCl) were vacuum filteredonto 1 μm polycarbonate filters that were immediately placed into 2 mLmicrofuge tubes containing 1.2 mL of 0.2M NaOH/2% SDS in distilled H₂O.After overnight lysis, the filters were removed from the tubes and thesupernatant buffer assayed for hydroxyproline according to the method ofGrant, supra.

EXAMPLE 2A Effects of Salt Concentration on Transmembrane Transport

To determine the effects of salt concentration on transmembranetransport, an approach similar to Example 1 was taken. A saturatedculture of JM109 (F—) harboring plasmid pD4 (FIG. 48) growing in LuriaBroth (LB) containing 100 μg/ml ampicillin (Amp) was used to inoculate20 ml cultures of LB/Amp to an OD at 600 nm of 0.1 AU. The cultures weregrown with shaking at 37° C. to an OD 600 run between 0.7 and 1.0 AU.Cells were collected by centrifugation and washed with 10 ml of M9media. Each cell pellet was resuspended in 20 ml of M9/Amp mediasupplemented with 0.5% glucose and 100 μg/ml of all of the amino acidsexcept proline. Cultures were grown at 37° C. for 30 min. to depleteendogenous proline. After out-growth, NaCl was added to the indicatedconcentration, Hyp was added to 40 mM, and IPTG to 1.5 mM. After 3 hoursat 37° C., cells from three 5 ml aliquots of each culture were collectedseparately on polycarbonate filters and washed twice with five ml of M9media containing 0.5% glucose and the appropriate concentration of NaCl.Cells were lysed in 1 ml of 70% ethanol by vortexing for 30 min. at roomtemperature. Cell lysis supernatants were taken to dryness, resuspendedin 100 μl of 2.5 N NaOH, and assayed for Hyp by the method of Neuman andLogan, R. E. Neuman and M. A. Logan, Journal of Biological Chemistry,184:299 (1950). Total protein was determined with the BCA kit (Pierce,Rockford Ill.) after cell lysis by three sonication/freeze-thaw cycles.The data are the mean±standard error of three separate experiments. Theintracellular concentration of trans-4-hydroxyproline versus NaClconcentration is illustrated graphically in FIG. 2A.

EXAMPLE 3 Determination of Proline Starvation Conditions in E. Coli

Proline auxotrophic E. coli strain NM519 (pro⁻) including plasmidpMAL-c2 which confers ampicillin resistance was grown in M9 minimalmedium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 μg mL ampicillinsupplemented with all amino acids at 20 μg/mL except proline which wassupplemented at 12.5 mg/L) to a constant AU₆₀₀ of 0.53 AU (17 hourspost-inoculation). Hydroxyproline was added to 0.08M andhydroxyproline-dependent growth was demonstrated by the increase in theOD₆₀₀ to 0.61 AU over a one hour period.

EXAMPLE 4 Hydroxyproline Incorporation Into Protein in E. coli UnderProline Starvation Conditions

Plasmid pMAL-c2 (commercially available from New England Biolabs)containing DNA encoding for maltose-binding protein (MBP) was used totransform proline auxotrophic E. coli strain NM519 (pro⁻). Two 1 Lcultures of transformed NM519 (pro⁻) in M9 minimal medium (M9 salts, 2%glucose, 0.01 mg/mL thiamine, 100 μg/mL ampicillin supplemented with allamino acids at 20 μg/mL except proline which was supplemented at 12.5mg/L) were grown to an AU₆₀₀ Of 0.53 (˜17 hours post-inoculation). Thecells were harvested by centrifugation, the media in one culture wasreplaced with an equal volume of M9 media containing 0.08Mhydroxyproline and the media in the second culture was replaced with anequal volume of M9 media containing 0.08M hydroxyproline and 0.5M NaCl.After a one hour equilibration, the cultures were induced with 1 mMisopropyl-β-D-thiogalactopyranoside. After growing for an additional3.25 hours, cells were harvested by centrifugation, resuspended in 10 mLof 10 mM Tris-HCl (pH 8), 1 mM EDTA, 100 mM NaCl (TEN buffer), and lysedby freezing and sonication. MBP was purified by passing the lysates over4 mL amylose resin spin columns, washing the columns with 10 mL of TENbuffer, followed by elution of bound MBP with 2 mL of TEN buffercontaining 10 mM maltose. Eluted samples were sealed in ampules undernitrogen with an equal volume of concentrated HCl (11.7M) and hydrolysedfor 12 hours at 120° C. After clarification with activated charcoal,hydroxyproline content in the samples was determined by HPLC and themethod of Grant, supra. The percent incorporation oftrans-4-hydroxyproline compared to proline into MBP is shown graphicallyin FIG. 12.

EXAMPLE 5 Hydroxyproline Incorporation Into Protein in S. cerevisiae ViaIntegrating Vectors Under Proline Starvation Conditions

The procedure described in Example 4 above is performed in yeast usingan integrating vector which disrupts the proline biosynthetic pathway. Agene encoding human Type 1 (α₁) collagen is inserted into a uniqueshuttle vector behind the inducible GALIO promoter. This promoter/genecassette is flanked by a 5′ and 3′ terminal sequence derived from a S.cerevisiae proline synthetase gene. The plasmid is linearized byrestriction digestion in both the 5′ and 3′ terminal regions and used totransform a proline-prototrophic S. cerevisiae strain. Thetransformation mixture is plated onto selectable media and transformantsare selected. By homologous recombination and gene disruption, theconstruct simultaneously forms a stable integration and converts the S.cerevisiae strain into a proline auxotroph. A single transformant isselected and grown at 30° C. in YPD media to an OD₆₀₀ of 2 AU. Theculture is centrifuged and the cells resuspended in yeast dropout mediasupplemented with all amino acids except proline and grown to a constantOD₆₀₀ indicating proline starvation conditions. 0.08M L-hydroxyprolineand 2% (w/v) galactose is then added. Cultures are grown for anadditional 648 hours. Cells are harvested by centrifugation (5000 rpm,10 minutes) and lysed by mechanical disruption.Hydroxyproline-containing human Type 1 (α₁) collagen is purified byammonium sulfate fractionation and column chromatography.

EXAMPLE 6 Hydroxyproline Incorporation Into Protein in S. cerevisiae ViaNon-Integrating Vectors Under Proline Starvation Conditions

The procedure described above in Example 4 is performed in a yeastproline auxotroph using a non-integrating vector. A gene encoding humanType 1 (α₁) collagen is inserted behind the inducible GAL10 promoter inthe YEp24 shuttle vector that contains the selectable Ura⁺ marker. Theresulting plasmid is transformed into proline auxotrophic S. cerevisiaeby spheroplast transformation. The transformation mixture is plated onselectable media and transformants are selected. A single transformantis grown at 30° C. in YPD media to an OD₆₀₀ of 2 AU. The culture iscentrifuged and the cells resuspended in yeast dropout mediasupplemented with all amino acids except proline and grown to a constantOD₆₀₀ indicating proline starvation conditions. 0.08M L-hydroxyprolineand 2% (w/v) galactose is then added. Cultures are grown for anadditional 6-48 hours. Cells are harvested by centrifugation (5000 rpm,10 minutes) and lysed by mechanical disruption.Hydroxyproline-containing human Type 1 (α₁) collagen is purified byammonium sulfate fractionation and column chromatography.

EXAMPLE 7 Hydroxyproline Incorporation Into Protein in a BaculovirusExpression System

A gene encoding human Type 1 (α₁) collagen is inserted into the pBacPAK8baculovirus expression vector behind the AcMNPV polyhedron promoter.This construct is co-transfected into SF9 cells along with linearizedAcMNPV DNA by standard calcium phosphate co-precipitation. Transfectantsare cultured for4 days at 27° C. in TNM-FH media supplemented with 10%FBS. The media is harvested and recombinant virus particles are isolatedby a plaque assay. Recombinant virus is used to infect 1 liter of SF9cells growing in Grace's media minus proline supplemented with 10% FBSand 0.08 M hydroxyproline. After growth at 27° C. for 2-10 days, cellsare harvested by centrifugation and lysed by mechanical disruption.Hydroxyproline-containing human Type 1 (α₁) collagen is purified byammonium sulfate fractionation and column chromatography.

EXAMPLE 8 Hydroxyproline Incorporation into Human Collagen Protein inEscherichia coli Under Proline Starvation Conditions

A plasmid (pHuCol, FIG. 4) encoding the gene sequence of human Type I(a,) collagen (FIGS. 3A and 3B) (SEQ. ID. NO. 1) placed behind theisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promotor andalso encoding β-lactamase is transformed into Escherichia coli prolineauxotrophic strain NM519 (pro⁻) by standard heat shock transformation.Transformation cultures are plated on Luria Broth (LB) containing 100μg/ml ampicillin and after overnight growth a singleampicillin-resistant colony is used to inoculate 5 ml of LB containing100 μg/ml ampicillin. After growth for 10-16 hours with shaking (225rpm) at 37° C., this culture is used to inoculate 1 L of M9 minimalmedium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 μg/mL ampicillin,supplemented with all amino acids at 20 μg/mL except proline which issupplemented at 12.5 mg/L) in a 1.5 L shaker flask. After growth at 37°C., 225 rpm, for 15-20 hours post-inoculation, the optical density at600 nm is constant at approximately 0.5 OD/nL. The cells are harvestedby centrifugation (5000 rpm, 5 minutes), the media decanted, and thecells resuspended in 1 L of M9 minimal media containing 100 μg/mLampicillin, 0.08M L-hydroxyproline, and 0.5M NaCl. Following growth for1 hour at 37° C., 225 rpm, IPTG is added to 1 mM and the culturesallowed to grow for an additional 5-15 hours. Cells are harvested bycentrifugation (5000-rpm, 10 minutes) and lysed by mechanicaldisruption. Hydroxyproline-containing collagen is purified by ammoniumsulfate fractionation and column chromatography.

Hydroxyproline Incorporation into Fragments of Human Collagen Protein inEscherichia coli Under Proline Starvation Conditions

A plasmid (pHuCol-F1, FIG. 6) encoding the gene sequence of the first 80amino acids of human Type 1 (α₁) collagen (FIG. 5) (SEQ. ID. NO. 2)placed behind the isopropyl-β-D-thiogalactopyranoside (IPTG)-inducibletac promotor and also encoding β-lactamase is transformed intoEscherichia coli proline auxotrophic strain NM519 (pro⁻) by standardheat shock transformation. Transformation cultures are plated on LuriaBroth (LB) containing 100 μg/mL ampicillin and after overnight growth asingle ampicillin-resistant colony is used to inoculate 5 mL of LBcontaining 100 μg/mL ampicillin. After growth for 10-16 hours withshaking (225 rpm) at 37° C., this culture is used to inoculate 1 L of M9minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 μg/mLampicillin, supplemented with all amino acids at 20 μg/mL except prolinewhich is supplemented at 12.5 mg/L) in a 1.5 L shaker flask. Aftergrowth at 37° C., 225 rpm, for 15-20 hours post-inoculation, the opticaldensity at 600 nm is constant at approximately 0.5 OD/mL. The cells areharvested by centrifugation (5000 rpm, 5 minutes), the media decanted,and the cells resuspended in 1 L of M9 minimal media containing 100μg/mL ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCl. Followinggrowth for 1 hour at 37° C., 225 rpm, IPTG is added to 1 mM and thecultures allowed to grow for an additional 5-15 hours. Cells areharvested by centrifugation (5000 rpm, 10 minutes) and lysed bymechanical disruption. The hydroxyproline-containing collagen fragmentis purified by ammonium sulfate fractionation and column chromatography.

EXAMPLE 10 Construction and Expression in E. coli of the Human CollagenType 1 (α₁) Gene with Optimized E. coli Codon Usage

A. Construction of the Gene:

The nucleotide sequence of the helical region of human collagen Type I(α₁) gene flanked by 17 amino acids of the amino terminal extra-helicaland 26 amino acids of the C-terminal extra-helical region is shown inFIG. 27 (SEQ. ID. NO. 15). A tabulation of the codon frequency of thisgene is given in Table I. The gene sequence shown in FIG. 27 was firstchanged to reflect E. coli codon bias. An initiating methionine wasinserted at the 5′ end of the gene and a TAAT stop sequence at the 3′end. Unique restriction sites were identified or created approximatelyevery 150 base pairs. The resulting gene (HuCol^(EC), FIG. 39A-39E)(SEQ. ID. NO. 20) has the codon usage given in Table II as shown below.Other sequences that approximate E. coli codon bias are also acceptable.TABLE II Codon Count % age Codon Count % age TTT-Phe 6 0.56 TCT-Ser 30.28 TTC-Phe 9 0.86 TCC-Ser 3 0.28 TTA-Leu 0 0.00 TCA-Ser 0 0.00 TTG-Leu0 0.00 TCG-Ser 0 0.00 CTT-Leu 0 0.00 CCT-Pro 13 1.22 CTC-Leu 1 0.09CCC-Pro 12 1.13 CTA-Leu 1 0.09 CCA-Pro 29 2.74 CTG-Leu 19 1.79 CCG-Pro18.6 17.58 ATT-Ile 3 0.28 ACT-Thr 2 0.18 ATC-Ile 4 0.37 ACC-Thr 11 1.03ATA-Ile 0 0.00 ACA-Thr 0 0.00 ATG-Met 8 0.75 ACG-Thr 4 0.37 GTT-Val 30.28 GCT-Ala 10 0.94 GTC-Val 5 0.47 GCC-Ala 24 2.26 GTA-Val 0 0.00GCA-Ala 8 0.75 GTG-Val 12 1.13 GCG-Ala 80 7.56 TAT-Tyr 2 0.18 TGT-Cys 00.00 TAC-Tyr 2 0.18 TGC-Cys 0 0.00 TAA-*** 0 0.00 TGA-*** 0 0.00 TAG-***0 0.00 TGG-Trp 0 0.00 CAT-His 0 0.00 CGT-Arg 26 2.45 CAC-His 3 0.28CGC-Arg 26 2.45 CAA-Gln 5 0.47 CGA-Arg 0 0.00 CAG-Gln 25 2.36 CGG-Arg 10.09 AAT-Asn 0 0.00 AGT-Ser 1 0.09 AAC-Asn 11 1.03 AGC-Ser 32 3.02AAA-Lys 38 3.59 AGA-Arg 0 0.00 AAG-Lys 0 0.00 AGG-Arg 0 0.00 GAT-Asp 201.89 GGT-Gly 14.8 13.98 GAC-Asp 14 1.32 GGC-Gly 17.8 16.82 GAA-Glu 403.78 GGA-Gly 9 0.85 GAG-Glu 9 0.85 GGG-Gly 12 1.13

Oligos of approximately 80 nucleotides were synthesized on a BeckmanOligo 1000 DNA synthesizer, cleaved and deprotected with aqueous NH₄OH,and purified by electrophoresis in 7M urea/12% polyacrylamide gels. Eachset of oligos was designed to have an EcoR I restriction enzyme site atthe 5′ end,-a unique restriction site near the 3′ end, followed by theTAAT stop sequence and a Hind III restriction enzyme site at the very 3′end. The first four oligos, comprising the first 81 amino acids of thehuman collagen Type I (α₁) gene, are given in FIG. 40 which shows thesequence and restriction maps of synthetic oligos used to construct thefirst 243 base pairs of the human Type I (α₁) collagen gene withoptimized E. coli codon usage. Oligos N1-1 (SEQ. ID. NO. 21) and N1-2(SEQ. ID. NO. 22) were designed to insert an initiating methionine (ATG)codon at the 5′ end of the gene.

In one instance, oligos N1-1 and N1-2 (1 μg each) were annealed in 20 μLof T7 DNA polymerase buffer (40 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 5mMdithiothreitol, 50 mM NaCl, 0.05 mg/mL bovine serum albumin) by heatingat 90° C. for 5 minutes followed by slow cooling to room temperature.After brief centrifugation at 14,000 rpm, 10 units of T7 DNA polymeraseand 2 μL of a solution of all four dNTPs (DATP, dGTP, dCTP, dTTP, 2.5 mMeach) were added to the annealed oligos. Extension reactions wereincubated at 37° C. for 30 minutes and then heated at 70° C. for 10minutes. After cooling to room temperature, Hind III buffer (5 μL of 10×concentration), 20 μL of H₂O, and 10 units of Hind III restrictionenzyme were added and the tubes incubated at 37° C. for 10 hours. HindIII buffer (2μL of 10× concentration), 13.5 μL of 0.5 M Tris.HCl (pH7.5), 1.8 μL of 1% Triton X100, 5.6 μL of H₂0, and 20 U of EcoR I wereadded to each tube and incubation continued for 2 hours at 37° C.Digests were extracted once with an equal volume of phenol, once withphenol/chloroform/isoamyl alcohol, and once with chloroform/isoamylalcohol. After ethanol precipitation, the pellet was resuspended in 10μL of TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Resuspended pellet(4 μL) was ligated overnight at 16° C. with agarose gel-purifiedEcoRI/Hind III digested pBSKS⁺ vector (1 μg) using T4 DNA ligase (100units). One half of the transformation mixture was transformed by heatshock into DH5α cells and 100 μL of the 1.0 mL transformation mixturewas plated on Luria Broth (LB) agar plates containing 70 μg/mLampicillin. Plates were incubated overnight at 37° C. Ampicillinresistant colonies (6-12) were picked and grown overnight in LB mediacontaining 70 mg/mL ampicillin. Plasmid DNA was isolated from eachculture by Wizard Minipreps (Promega Corporation, Madison Wis.) andscreened for the presence of the approximately 120 base pair insert bydigestion with EcoR I and Hind III and running the digestion products onagarose electrophoresis gels. Clones with inserts were confirmed bystandard dideoxy termination DNA sequencing. The correct clone was namedpBSN1-1 (FIG. 41) and the collagen fragment has the nucleic acidsequence given in FIG. 42 (SEQ. ID. NO. 25).

Oligos N1-3 (SEQ. ID. NO. 23) and N1-4 (SEQ. ID. NO. 24) (FIG. 40) weresynthesized, purified, annealed, extended, and cloned into pBSKS⁺following the same procedure given above for oligos N1-1 and N1-2. Theresulting plasmid was named pBSN1-2A. To clone together the sections ofthe collagen gene from pBSN1-1 and pBSN1-2A, plasmid pBSN1-1 (1 μg) wasdigested for 2 hours at 37° C. with Rsr II and Hind III. The digestedvector was purified by agarose gel electrophoresis. Plasmid pBSN1-2A (3μg) was digested for 2 hours at 37° C. with Rsr II and Hind III and theinsert purified by agarose gel electrophoresis. Rsr II/Hind III-digestedpBSN1-1 was ligated with this insert overnight at 16° C. with T4 DNAligase. One half of the ligation mixture was transformed into DH5α cellsand 1/10 of the transformation mixture was plated on LB agar platescontaining 70 μg/mL ampicillin. After overnight incubation at 37° C.,ampicillin-resistant clones were picked and screened for the presence ofinsert DNA as described above. Clones were confirmed by dideoxytermination sequencing. The correct clone was named pBSN1-2 (FIG. 43)and the collagen fragment has the sequence given in FIG. 44.

In similar manner, the remainder of the collagen gene is constructedsuch that the final DNA sequence is that given in FIG. 39A-39E (SEQ. ID.NO. 19).

B) Expression of the Gene in E. coli:

Following construction of the entire human collagen Type I (α₁) genewith codon usage optimized for E. coli, the cloned gene is expressed inE. coli. A plasmid (pHuCol^(Ec), FIG. 45) encoding the entire syntheticcollagen gene (FIG. 39A-39E) placed behind theisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promotor andalso encoding β-lactamase is transformed into Escherichia coli strainDH5α (supE44 ΔlacU169 (φ80lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1relA1) by standard heat shock transformation. Transformation culturesare plated on Luria Broth (LB) containing 100 μg/mL ampicillin and afterovernight growth a single ampicillin-resistant colony is used toinoculate 10 mL of LB containing 100 μg/mL ampicillin. After growth for10-16 hours with shaking (225 rpm) at 37° C., this culture is used toinoculate 1 L of LB containing 100 μg/mL ampicillin in a 1.5 L shakerflask. After growth at 37° C., 225 rpm, for 2 hours post-inoculation,the optical density at 600 nm is approximately 0.5 OD/nL. IPTG is addedto 1 mM and the culture allowed to grow for an additional 5-10 hours.Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysedby mechanical disruption. Recombinant human collagen is purified byammonium sulfate fractionation and column chromatography. The yield istypically 15-25 mg/L of culture.

EXAMPLE 11 Expression in E. coli of an 81 Amino Acid Fragment of HumanCollagen Type I(α1) with Optimized E. coli Codon Usage

A plasmid (pTrcN1-2, FIG. 46) encoding the gene sequence of the first 81amino acids of human Type I (α₁) collagen with optimized E. coli codonusage cloned in fusion with a 6 histidine tag at the 5′ end of the geneand-placed-behind the isopropyl-β-D-thiogalactopyranoside(IPTG)-inducible trc promotor and also encoding β-lactamase wasconstructed by subcloning the EcoR I/Hind III insert from pBSN1-2 intothe EcoR I/Hind III site of plasmid pTrcB (Invitrogen, San Diego,Calif.). Plasmid pTrcN1-2 was transformed into Escherichia coli strainDH5α (supE44ΔlacU169 (φ80lacIZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1relA1) by standard heat shock transformation. Transformation cultureswere plated on Luria Broth (LB) containing 100 μg/mL ampicillin andafter overnight growth a single ampicillin-resistant colony was used toinoculate 5 mL of LB containing 100 μg/mL ampicillin. After growth for10-16 hours with shaking (225 rpm) at 37° C., this culture was used toinoculate 50 mL of LB containing 100 μg/mL ampicillin in a 250 mL shakerflask. After growth at 37° C., 225 rpm, for 2 hours post-inoculation,the optical density at 600 nm was approximately 0.5 OD/mL. IPTG wasadded to 1 mM and the culture allowed to grow for an additional 5-10hours. Cells were harvested by centrifugation (5000 rpm, 10 minutes) andstored at −20° C. The 6 histidine tag-collagen fragment fusion waspurified on nickel resin columns. Cell pellets were resuspended in 10 mLof 6M guanidine hydrochloride/20 mM sodium phosphate/500 mM NaCl (pH7.8) and bound in two 5 mL batches to the nickel resin. Columns werewashed two times with 4 mL of binding buffer (8M urea/20 mMsodium-phosphate/500 mM NaCl (pH 7.8)), two times with wash buffer 1 (8Murea/20 mM sodium phosphate/500 mM NaCl (pH 6.0)), and two times withwash buffer 2 (8 m urea/20 mM sodium phosphate/500 mM NaCl (pH 5.3). The6 histidine tag-collagen fragment fusion was eluted from the column with5 mL of elution buffer (8M urea/20 mM sodium phosphate/500 mM NaCl (pH4.0) in 1 mL fractions. Fractions were assessed for protein by gelelectrophoresis and fusion-containing fractions were concentrated andstored at −20° C. The yield was typically 15-25 mg/L of culture.

The collagen is cleaved from the 6 histidine tag with enterokinase.Fusion-containing fractions are dialyzed against cleavage buffer (50 mMTris-HCl, pH 8.0/5 mM CaCl₂). After addition of enterokinase at 1 μgenzyme for each 100 μg fusion, the solution is incubated at 37° C. for4-10 hours. Progress of the cleavage is monitored by gelelectrophoresis. The cleaved 6 histidine tag may be separated from thecollagen fragment by passage over a nickel resin column as outlinedabove.

EXAMPLE 12 Expression in E. coli of Fragments of Human Collagen Type I(α₁) with Optimized E. coli Codon Usage

A plasmid (pN 1-3, FIG. 47) encoding the gene for the amino terminal 120amino acids of human collagen Type I (α₁) with optimized E. coli codonusage placed behind the isopropyl-β-D-thiogalactopyrano side(IPTG)-inducible tac promotor and also encoding β-lactamase istransformed into Escherichia coli strain DH5a (sup E44 ΔlacU169 (φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) by standard heat shocktransformation. Transformation cultures are plated on Luria Broth (LB)containing 100 μg/mL ampicillin and after overnight growth a singleampicillin-resistant colony is used to inoculate 10 mL of LB containing100 μg/mL ampicillin. After growth for 10-16 hours with shaking (225rpm) at 37° C., this culture is used to inoculate 1 L of LB containing100 μg/mL ampicillin in a 1.5 L shaker flask. After growth at 37° C.,225 rpm, for 2 hours post-inoculation, the optical density at 600 nm isapproximately 0.5 OD/mL. IPTG is added to 1 mM and the culture allowedto grow for an additional 5-10 hours. Cells are harvested bycentrifugation (5000 rpm, 10 minutes) and lysed by mechanicaldisruption. Recombinant human collagen is purified by ammonium sulfatefractionation and column chromatography. The yield is typically 15-25mg/L of culture.

EXAMPLE 13 Expression in E. coli of a C-terminal Fragment of HumanCollagen Type I (α₁) with Optimized E. coli Codon Usage

A plasmid (pD4, FIG. 48) encoding the gene for the carboxy terminal 219amino acids of human collagen Type I (α₁) with optimized E. coli codonusage placed behind the isopropyl-β-D-thiogalactopyranoside(IPTG)-inducible tac promotor and also encoding β-lactamase istransformed into Escherichia coli strain DH5α (sup E44 ΔlacU169 (φ80lacZΔM15) hsdR17 recA1 endA1 grA96 thi-1 relA1) by standard heat shocktransformation. Transformation cultures are plated on Luria Broth (LB)containing 100 μg/mL ampicillin and after overnight growth a singleampicillin-resistant colony is used to inoculate 10 mL of LB containing100 μg/mL ampicillin. After growth for 10-16 hours with shaking (225rpm) at 37° C., this culture is used to inoculate 1 L of LB containing100 μg/mL ampicillin in a 1.5 L shaker flask. After growth at 37° C.,225 rpm, for 2 hours post-inoculation, the optical density at 600 nm isapproximately 0.5 OD/mL. IPTG is added to 1 mM and the culture allowedto grow for an additional 5-10 hours. Cells are harvested bycentrifugation (5000 rmp, 10 minutes) and lysed by mechanicaldisruption. Recombinant human collagen fragment is purified by ammoniumsulfate fractionation and column chromatography. The yield is typically15-25 mg/L of culture.

EXAMPLE 14 Construction and Expression in E. coli of the Human CollagenType 1 (α2) Gene with Optimized E. coli Codon Usage

A) Construction of the Gene:

The nucleotide sequence of the helical region of human collagen Type I(α2) gene flanked by 11 amino acids of the amino terminal extra-helicaland 12 amino acids of the C-terminal extra-helical region is shown inFIGS. 49A-49E (SEQ. ID. NO. 29). A tabulation of the codon frequency ofthis gene is given in Table III below. The gene sequence shown in FIGS.49A-49E was first changed to reflect E. coli codon bias. An initiatingmethionine was inserted at the 5′ end of the gene and a TAAT stopsequence at the 3′ end. Unique restriction sites are identified orcreated approximately every 150 base pairs. The resulting gene(HuCol(α₂)^(Ec), FIGS. 50A-50E) (SEQ. ID. NO. 31) has the codon usagegiven in Table IV below. Other sequences that approximate E. coli codonbias are also acceptable. TABLE III Codon Count % age Codon Count % ageTTT-Phe 3 0.28 TCT-Ser 11 1.06 TTC-Phe 10 0.96 TCC-Ser 4 0.38 TTA-Leu 10.09 TCA-Ser 1 0.09 TTG-Leu 2 0.19 TCG-Ser 1 0.09 CTT-Leu 16 1.54CCT-Pro 125 12.06 CTC-Leu 9 0.86 CCC-Pro 42 4.05 CTA-Leu 2 0.19 CCA-Pro30 2.89 CTG-Leu 5 0.48 CCG-Pro 3 0.28 ATT-Ile 14 1.35 ACT-Thr 14 1.35ATC-Ile 3 0.28 ACC-Thr 0 0.00 ATA-Ile 1 0.09 ACA-Thr 3 0.28 ATG-Met 50.48 ACG-Thr 1 0.09 GTT-Val 20 1.93 GCT-Ala 82 7.91 GTC-Val 5 0.48GCC-Ala 17 1.64 GTA-Val 3 0.28 GCA-Ala 9 0.86 GTG-Val 10 0.96 GCG-Ala 00.00 TAT-Tyr 2 0.19 TGT-Cys 0 0.00 TAC-Tyr 3 0.28 TGC-Cys 0 0.00 TAA-***0 0.00 TGA-*** 0 0.00 TAG-*** 0 0.00 TGG-Trp 0 0.00 CAT-His 7 0.67CGT-Arg 17 1.64 CAC-His 6 0.57 CGC-Arg 6 0.57 CAA-Gln 13 1.25 CGA-Arg 60.57 CAG-Gln 9 0.86 CGG-Arg 4 0.38 AAT-Asn 10 0.96 AGT-Ser 11 1.06AAC-Asn 14 1.35 AGC-Ser 4 0.38 AAA-Lys 15 1.44 AGA-Arg 16 1.54 AAG-Lys16 1.54 AGG-Arg 6 0.57 GAT-Asp 20 1.93 GGT-Gly 179 17.27 GAC-Asp 5 0.48GGC-Gly 74 7.14 GAA-Glu 29 2.79 GGA-Gly 80 7.72 GAG-Glu 16 1.54 GGG-Gly16 1.54

TABLE IV Codon Count % age Codon Count % age TTT-Phe 5 0.48 TCT-Ser 70.67 TTC-Phe 7 0.67 TCC-Ser 12 1.15 TTA-Leu 0 0.00 TCA-Ser 0 0.00TTG-Leu 0 0.00 TCG-Ser 0 0.00 CTT-Leu 1 0.09 CCT-Pro 10 0.96 CTC-Leu 10.09 CCC-Pro 0 0.00 CTA-Leu 0 0.00 CCA-Pro 15 1.44 CTG-Leu 32 3.07CCG-Pro 17.7 17.00 ATT-Ile 11 1.05 ACT-Thr 3 0.28 ATC-Ile 7 0.67 ACC-Thr6 0.57 ATA-Ile 0 0.00 ACA-Thr 0 0.00 ATG-Met 6 0.57 ACG-Thr 10 0.96GTT-Val 18 1.72 GCT-Ala 30 2.88 GTC-Val 7 0.67 GCC-Ala 21 2.01 GTA-Val 90.86 GCA-Ala 20 1.92 GTG-Val 6 0.57 GCG-Ala 38 3.65 TAT-Tyr 3 0.28TGT-Cys 0 0.00 TAC-Tyr 2 0.19 TGC-Cys 0 0.00 TAA-*** 0 0.00 TGA-*** 00.00 TAG-*** 0 0.00 TGG-Trp 0 0.00 CAT-His 2 0.19 CGT-Arg 37 3.56CAC-His 11 1.05 CGC-Arg 18 1.72 CAA-Gln 7 0.67 CGA-Arg 0 0.00 CAG-Gln 151.44 CGG-Arg 0 0.00 AAT-Asn 6 0.57 AGT-Ser 0 0.00 AAC-Asn 18 1.72AGC-Ser 13 1.24 AAA-Lys 25 2.40 AGA-Arg 0 0.00 AAG-Lys 6 0.57 AGG-Arg 00.00 GAT-Asp 11 1.06 GGT-Gly 20.9 20.07 GAC-Asp 13 1.24 GGC-Gly 14.113.54 GAA-Glu 33 3.17 GGA-Gly 0 0.00 GAG-Glu 12 1.15 GGG-Gly 0 0.00

Oligos of approximately 80 nucleotides are synthesized on a BeckmanOligo 1000 DNA synthesizer, cleaved and deprotected with aqueous NH₄OH,and purified by electrophoresis in 7M urea/12% polyacrylarnide gels.Each set of oligos is designed to have an EcoR I restriction enzyme siteat the 5′ end, a unique restriction site near the 3′ end, followed bythe TAAT stop sequence and a Hind III restriction enzyme site at thevery 3′ end. Oligos N1-1(α₂) and N1-2(α2) are designed to insert aninitiating methionine (ATG) codon at the 5′ end of the gene.

In one instance, oligos N1-1(α₂) and N1-2(α₂) (1 μg each) (FIG. 51depicts sequence and restriction maps of synthetic oligos used toconstruct the first 240 base pairs of human Type I (α2) collagen genewith optimized E. coli codon usage) are annealed in 20 μL of T7 DNApolymerase buffer (40 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 5 mMdithiothreitol, 50 mM NaCl, 0.05 mg/mL bovine serum albumin) by heatingat 90° C. for 5 minutes followed by slow cooling to room temperature.After brief centrifugation at 14,000 rpm, 10 units of T7 DNA polymeraseand 2 μL of a solution of all four dNTPs (DATP, dGTP, dCTP, dTTP, 2.5 mMeach) are added to the annealed oligos. Extension reactions areincubated at 37° C. for 30 minutes and then heated at 70° C. for 10minutes. After cooling to room temperature, Hind III buffer (5 μL of 10×concentration), 20 μL of H₂O, and 10 units of Hind III restrictionenzyme are added and the tubes incubated at 37° C. for 10-16 hours. HindIII buffer (2 IL of 10× concentration), 13.5 μL of 0.5 Tris-HCl (pH7.5), 1.8 μL of 1% Triton X100, 5.6 μL of H₂O, and 20 U of EcoR I areadded to each tube and incubation continued for 2 hours at 37° C.Digests are extracted once with an equal volume of phenol, once withphenol/chloroform/isoamyl alcohol, and once with chloroform/isoamylalcohol. After ethanol precipitation, the pellet is resuspended in 10 μLof TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Resuspended pellet (4μL) is ligated overnight at 16° C. with agarose gel-purified EcoRI/HindIII digested pBSKS⁺ vector (1 μg) using T4 DNA ligase (100 units). Onehalf of the transformation mixture is transformed by heat shock intoDH5α cells and 100 μL of the 1.0 mL transformation mixture is plated onLuria Broth (LB) agar plates containing 70 μg/mL ampicillin. Plates areincubated overnight at 37° C. Ampicillin resistant colonies (6-12) arepicked and grown overnight in LB media containing 70 μg/mL ampicillin.Plasmid DNA is isolated from each culture by Wizard Minipreps (PromegaCorporation, Madison, Wis.) and screened for the presence of theapproximately 120 base pair insert by digestion with EcoR I and Hind IIIand running the digestion products on agarose electrophoresis gels.Clones with inserts are confirmed by standard dideoxy termination DNAsequencing. The correct clone is named pBSN1-1 (α2) FIG. 52).

Oligos N1-3(α2) and N-4(α2) are synthesized, purified, annealed,extended, and cloned into pBSKS⁺ following the same procedure givenabove for oligos N1-1(α₂) and N1-2(α2). The resulting plasmid is namedpBSN1-2A. To clone together the sections of the collagen gene frompBSN1-1(α₂) (1 μg) is digested for 2 hours at 37° C. with BsrF I andHind III. The digested vector is purified by agarose gelelectrophoresis. Plasmid pBSn1-2(α₂) (3 μg) is digested for 2 hours at37° C. with BsrF I and Hind III and the insert purified by agarose gelelectrophoresis. BsrF I/Hind III-digested pBSN1-1 is ligated with thisinsert overnight at 16° C. with T4 DNA ligase. One half of the ligationmixture is transformed into DH5α cells and 1/10 of the transformationmixture is plated on LB agar plates containing 70 μg/mL ampicillin.After overnight incubation at 37° C., ampicillin-resistant clones arepicked and screened for the presence of insert DNA as described above.Clones are confirmed by dideoxy termination sequencing. The correctclone is name pBSN1-2(α2) (FIG. 53) and the collagen fragment has thesequence given in FIG. 54 (SEQ. ID. NO. 37).

In a similar manner, the remainder of the collagen gene is constructedsuch that the final DNA sequence is that given in FIGS. 50A-50E (SEQ.ID. NO.31).

B) Expression of the Gene in E. coli:

Following construction of the entire human collagen Type I (α2) genewith codon usage optimized for E. coli, the cloned gene is expressed inE. coli. A plasmid (pHuCol(α₂)^(Ec), FIG. 55) encoding the entiresynthetic collagen gene (FIGS. 50A-50E) placed behind theisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promotor andalso encoding β-lactamase is transformed into Escherichia coli strainDH5α (supE44 ΔlacU169 (φ80lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1relA1) by standard heat shock transformation. Transformation culturesare plated on Luria Broth (LB) containing 100 μg/mL ampicillin and afterovernight growth a single ampicillin-resistant colony is used toinoculate 10 mL of LB containing 100 μg/mL ampicillin and afterovernight growth a single ampicillin-resistant colony is used toinoculate 10 mL of LB containing 100 μg/mL ampicillin. After growth for10-16 hours with shaking (225 rpm) at 37° C., this culture is used toinoculate 1 L of LB containing 100 μg/mL ampicillin in a 1.5 L shakerflask. After growth at 37° C., 225 rpm, for 2 hours post-inoculation,the optical density at 600 nm is approximately. 0.5 OD/mL. IPTG is addedto 1 mM and the culture allowed to grow for an additional 5-10 hours.Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysedby mechanical disruption. Recombinant human collagen is purified byammonium sulfate fractionation and column chromatography. The yield istypically 15-25 mg/L of culture.

EXAMPLE 14A Alternative Construction and Expression in E. Coli of theHuman Collagen Type 1 (α2) Gene with Optimized E. coli Codon Usage

A) Construction of the Gene:

The nucleotide sequence of the helical region of human collagen Type 1(α2) gene flanked by 11 amino acids of the amino terminal extra-helicaland 12 amino acids of the C-terminal extra-helical region is shown inFIGS. 49A-49E (SEQ. ID. NO. 29). A tabulation of the codon frequency ofthis gene is given in Table III. The gene sequence shown in FIGS.49A-49E was first changed to reflect E. coli codon bias. An initiatingmethionine was inserted at the 5′ end of the gene and a TAAT stopsequence at the 3′ end. Unique restriction sites were identified orcreated at appropriate locations in the gene (approximately every 150base pairs). The resulting gene (HuCol(α2)^(Ec), FIGS. 50A-50E) (SEQ.ID. NO. 31) has the codon usage given in Table IV. Other sequences thatapproximate E. coli codon bias are also acceptable.

Oligonucleotides were synthesized on a Beckman Oligo 1000 DNAsynthesizer, cleaved and deprotected with aqueous NH₄OH, and purified byelectrophoresis in 7M urea/12% polyacrylamide gels. Purified oligos(32.5 pmol) were dissolved in 20 μL of ligation buffer (BoehringerMannheim, Cat. No. 1635 379) and annealed by heating to 95° C. followedby slow cooling to 20° C. over 45 minutes. The annealed oligonucleotideswere ligated for 5 minutes at room temperature with digested vector (1μg) using T4 DNA ligase (5 units). One half of the transformationmixture was transformed by heat shock into DH5α cells and 100 μL of the1.0 mL transformation mixture plated on Luria Broth (LB) agar platescontaining 70 mg/mL ampicillin. Plates were incubated overnight at 37°C. Ampicillin resistant colonies (6-12) were picked and grown overnightin LB media containing 70 μg/mL ampicillin. Plasmid DNA was isolatedfrom each culture by QIAprep Miniprep (Qiagen, Valencia, Calif.) andscreened for the presence of insert by digestion with flankingrestriction enzymes and running the digestion products on agaroseelectrophoresis gels. Clones with inserts were confirmed by standarddideoxy termination DNA sequencing. To clone together the sections ofthe collagen gene, and insert covering a flanking portion of the genewas ligated into vector containing the neighboring gene portion. Insertswere isolated from plasmids and vectors were cut by double digestion for2 hours at 37° C. with the appropriate restriction enzymes. The digestedvector and insert were purified by agarose gel electrophoresis. Insertand vector were ligated for 5 minutes at room temperature following theprocedure in the Rapid DNA Ligation Kit (Boehringer Mannheim). One halfof the ligation mixture is transformed into DH5α cells and 1/10 of thetransformation mixture was plated on LB agar plates containing 70 μg/mLampicillin. After overnight incubation at 37° C., ampicillin-resistantclones were picked and screened for the presence of insert DNA asdescribed above. Clones were confirmed by dideoxy terminationsequencing.

In a similar manner, the remainder of the collagen gene was constructedsuch that the final DNA sequence is that given in FIGS. 50A-50E (SEQ.ID. NO. 31).

B) Expression of the Gene in E. coli:

Following construction of the entire human collagen Type 1 (α2) genewith codon usage optimized for E. coli, the cloned gene is expressed inE. coli. A plasmid (pHuCol)(α2)^(Ec), FIG. 55) encoding the entirecollagen gene (FIGS. 50A-50E) placed behind theisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promoter andalso encoding β-lactamase is transformed into Escherichia coli strainDH5α (supE44 ΔlacU169 (φ80lacZ ΔM15) hsdR17 recA1 endA1 grA96 thi-1relA1) by standard heat shock transformation. Transformation culturesare plated on Luria Broth (LB) containing 100 μg/mL ampicillin and afterovernight growth a single ampicillin-resistant colony is used toinoculate 10 mL of LB containing 100 μg/mL ampicillin. After growth for10-16 hours with shaking (225 rpm) at 37° C., this culture is used toinoculate 1 L of LB containing 100 μg/mL ampicillin in a 1.5 L shakerflask. After growth at 37° C., 225 rpm, for 2 hours post-inoculation,the optical density at 600 nm is approximately 0.5 OD/mL. IPTG is addedto 1 mM and the culture allowed to grow for an additional 5-10 hours.Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysedby mechanical disruption. Recombinant human collagen is purified byammonium sulfate fractionation and column chromatograph. The yield istypically 15-25 mg/L of culture.

EXAMPLE 15 Expression in E. coli of Fragments of Human Collagen Type I(α2) with Optimized E. coli Codon Usage

A plasmid (pN 1-2, FIG. 56) encoding the gene for the amino terminal 80amino acids of human collagen Type I (α2) (SEQ. ID. NO. 31, FIG. 54)with optimized E. coli codon usage placed behind theisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible tac promotor andalso encoding P-lactamase is transformed into Escherichia coli strainDH5α (supE44 ΔlacU169 (φ80lacZ ΔM15) hsdr17 recA1 endA1 gyrA96 thi-1relA1) by standard heat shock transformation. Transformation culturesare plated on Luria Broth (LB) containing 100 μg/mL ampicillin and afterovernight growth a single ampicillin-resistant colony is used toinoculate 10 mL of LB containing 100 μg/mL ampicillin. After growth for10-16 hours with shaking (225 rpm) at 37° C., this culture is used toinoculate 1 L of LB containing 100 μg/mL ampicillin in a 1.5 L shakerflask. After growth at 37° C., 225 rpm, for 2 hours post-inoculation,the optical density at 600 nm is approximately 0.5 OD/mL. IPTG is addedto 1 mM and the culture allowed to grow for an additional 5-10 hours.Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysedby mechanical disruption. Recombinant human collagen is purified byammonium sulfate fractionation and column chromatography. The yield istypically 15-25 mg/L of culture.

EXAMPLE 16 Hydroxyproline Incorporation into Proteins in E. coli UnderProline Starvation Conditions

Seven plasmids, pGEX-4T.1 (FIG. 73), pTrc-TGF (FIG. 74), pMal-C2 (FIG.1), pTrc-FN (FIG. 75), pTrc-FN-TGF (FIG. 76), pTrc-FN-Bmp (FIG. 77) andpGEX-HuColl^(Ec), each separately containing genes encoding thefollowing proteins: glutathione S-transferase (GST), the mature humanTGF-β1 polypeptide (TGF-β1), mannose-binding protein (MBP), a 70 kDAfragment of human fibronectin (FN), a fusion of FN and TGF-β1(FN-TGF-β1), a fusion of FN and human bone morphogenic protein 2A(FN-BMP-2A), and a fusion of GST and collagen (GST-Coll), were usedindividually to transform proline auxotrophic E. coli strain JM109 (F—).Transformation cultures were plated on LB agar containing 100 μg/mlampicillin. After overnight incubation at 37° C., a single colony from afresh transformation plate was used to inoculate 5 ml of LB mediacontaining 400 mg ampicillin. After overnight growth at 37° C., thisculture was centrifuged, the supernatant discarded, and the cell pelletwashed twice with 5 ml of M9 medium (1×M9 salts, 0.5% glucose, 1 mMMgCl₂, 0.01% thiamine, 200 μg/ml glycine, 200 μg/ml alanine, 100 μg/mlof the other amino acids except proline, and 400 μg/ml ampicillin). Thecells were finally resuspended in 5 ml of M9 medium. After incubationwith shaking at 37° C. for 30 minutes, trans-4-hydroxyproline was addedto 40 nM, NaCl to 0.5 M, and isopropyl-B-D-thiogalactopyyranoside to 1.5mM. In certain cultures one of these additions was not made, asindicated in the labels for the lanes of the gels. After addition,incubation with shaking at 37° C. was continued. After 4 hours, thecultures were centrifuged, the supernatants discarded, and the cellpellets resuspended in SDS-PAGE sample buffer (300 mM Tris (pH6.8)/0.5%SDS/10% glycerol/0.4M P-mercapthoethanol/0.2% bromophenol blue) to 15OD600 nm AU/ml, placed in boiling water bath for five minutes, andelectrophoresed in denaturing polyacrylaminde gels. Proteins in the gelswere visualized by staining with Coomassie Blue R250. The results of thegels are depicted in scans shown in FIGS. 57-59. The scans relating toGST, TGF-β1, MBP, FN, FN-TGF-β1, and FN-BMP-2A (FIGS. 57 and 58) showthree lanes relating to each peptide, i.e., one lane indicating−NaCl/+Hyp wherein NaCl (hyperosmotic) and trans-4-hydroxyproline arepresent; one lane indicating —NaCl wherein trans-4-hydroxyproline ispresent but NaCl is not; and one lane indicating −Hyp which is +NaCl butabsent trans-4-hydroxyproline. Asterisks on the scans mark protein bandswhich correspond to the expressed target protein. The instances in whichtarget protein was expressed all involve +NaCl in connection with +Hypthus demonstrating +NaCl and +Hyp dependence.

The scan shown in FIG. 59 relating to GST-collagen shows four lanesrelating to GST-Coll, i.e., one lane indicating +Hyp/+NaCl/−IPTG whereintrans-4-hydroxyproline and NaCl are present but IPTG (the proteinexpression inducer) is not and since there is no inducer, there is notarget protein band; one lane indicating +NaCl/+IPTG/-Hyp wherein NaCland IPTG are present but trans-4-hydroxyproline is not and, sincetrans-4-hydroxyproline is not present no target protein band is evident;one lane indicating +NaCl/+Pro/+IPTG wherein NaCl, proline and IPTG arepresent, but since the target protein is not stable when it containsproline, there is no target protein band; and one lane designated+IPTG/+NaCl/+Hyp wherein IPTG, NaCl and trans-4-hydroxyproline arepresent and since the protein is stabilized by the presence oftrans-4-hydroxyproline an asterisk marked protein band is evident.

EXAMPLE 17 Hydroxyproline Incorporation into a Collagen-Like Peptide inE. coli

A plasmid (pGST-CM4, FIG. 60) containing the gene for collagen mimetic 4(CM4, FIG. 61) (SEQ. ID. NO. 39) genetically linked to the 3′ end of thegene for S. japonicum glutathione S-transferase was used to transform byelectroporation proline auxotrophic E. coli strain JM109 (F—).Transformation cultures were plated on LB agar containing 100 μg/mlampicillin. After overnight incubation at 37° C., a single colony from afresh transformation plate was used to inoculate 5 ml of LB mediacontaining 100 μg/ml ampicillin. After overnight growth at 37° C., 500μl of this culture was centrifuged, the supernatent discarded, and thecell pellet washed once with 500 μl of M9 medium (1×M9 salts, 0.5%glucose, 1 mM MgCl₂, 0.01% thiamine, 200 μg/ml glycine, 200 μg/mlalanine, 100 μg/ml of the other amino acids except proline, and 400μg/ml ampicillin). The cells were finally suspended in 5 ml of M9 mediumcontaining 10 μg/ml proline and 2 ml of this was used to inoculate 30 mlof M9 medium containing 10 μg/ml proline. After incubation with shakingat 37° C. for 8 hours, the culture was centrifuged and the cell pelletwashed once with M9 medium containing 5 μg/ml proline. The pellet wasresuspended in 15 ml of M9 medium containing 5 μg/ml of proline and thisculture was used to inoculate 1 L of M9 medium containing 5 μg/ml ofproline. This culture was grown for 18 hours at 37° C. to prolinestarvation. At this time, the culture was centrifuged, the cells washedonce with M9 medium (with no proline), and the cells resuspended in 1 Lof M9 medium containing 80 mM hydroxyproline, 0.5 M NaCl, and 1.5 mMisopropyl-β-D-thiogalactopyranoside. Incubation was continued at 37° C.with shaking for 22 hours. The cultures were centrifuged and the cellpellets stored at −20° C. until processed further.

EXAMPLE 18 Proline Incorporation into a Collagen-Like Peptide in E. coli

A plasmid (pGST-CM4, FIG. 60) containing the gene for collagen mimetic 4(CM4, FIG. 61) (SEQ. ID. NO. 39) genetically linked to the 3′ end of thegene for S. japonicum glutathione S-transferase was used to transform byelectroporation proline auxotrophic E. coli strain JM109 (F—).Transformation cultures were plated on LB agar containing 100 μg/mlampicillin. After overnight incubation at 37° C., a single colony from afresh transformation plate was used to inoculate 5 ml of LB mediacontaining 100 μg/ml ampicillin. After overnight growth at 37° C., 500μl of this culture was centrifuged, the supernatent discarded, and thecell pellet washed once with 500 μl of M9 medium (1×M9 salts, 0.5%glucose, 1 mM MgCl₂, 0.01% thiamine, 200 μg/ml glycine, 200 μg/mlalanine, 100 μg/ml of the other amino acids except proline, and 400μg/mL ampicillin). The cells were finally resuspended in 5 ml of M9medium containing 10 μg/ml proline and 2 ml of this was used toinoculate 30 ml of M9 medium containing 10 μg/ml proline. This culturewas incubated with shaking at 37° C. for 8 hours. The culture wascentrifuged and the cell pellet washed once with M9 medium containing 5μg/ml proline. The pellet was resuspended in 15 ml of M9 mediumcontaining 5 μg/ml of proline and this culture was used to inoculate 1 Lof M9 medium containing 5 μg/ml of proline. This culture was grown for18 hours at 37° C. to proline starvation. At this time, the culture wascentrifuged, the cells washed once with M9 medium (with no proline), andfinally the cells were resuspended in 1 L of M9 medium containing 2.5 mMproline, 0.5 M NaCl, and 1.5 mM isopropyl-p-p-thiogalactopyranoside.Incubation was continued at 37° C. with shaking for 22 hours. Thecultures were then centrifuged and the cell pellets stored at −20° C.until processed further.

EXAMPLE 19 Purification of Hydroxyproline-Containing Collagen-LikePeptide from E. coli

The cell pellet from a 1 L fermentation culture prepared as described inExample 17 above, was resuspended in 20 ml of Dulbecco's phosphatebuffered saline (pH 7.1) (PB1S) containing 1 mM EDTA, 100 μM PMSF, 0.5μg/ml E64, and 0.7 μg/ml pepstatin (resuspension buffer). The cells werelysed by twice passing through a French press. Following lysis, thesuspension was centrifuged for 30 minutes at 30,000×g. The supernatentwas discarded and the pellet washed once with 5 ml of resuspensionbuffer containing 1 M urea and 0.5% Triton X100 followed by one washwith 7 ml of resuspension buffer without urea or Triton X100. The pelletwas finally resuspended in 5 ml of 6M guanidine hydrochloride inDulbecco's phosphate buffered saline (pH7.1) containing 1 mM EDTA and 2mM β-mercaptoethanol and sonicated on ice for 3×60 seconds (microtip,power=3.5, Heat Systems XL-2020 model sonicator). The sonicatedsuspension was incubated at 4° C. for 18 hours and then centrifuged at14,000 rpm in a microcentrifuge. The supernatent (6 ml) was dialyzed(10,000 MWCO) against 4×4 L of distilled water at 4° C. The contents ofthe dialysis tubing were transferred to a 150 ml round bottom flask andlyophilized to dryness. The residue (˜30 mg) was dissolved in 3 ml of70% formic acid and 40 mg of cyanogen bromide was added. The flask wasflushed once with nitrogen, evacuated, and allowed to stir for 18 hoursat room temperature. The contents of the flask were taken to dryness invacuo at room temperature, the residue resuspended in 5 ml of distilledwater and evaporated to dryness again. This was repeated 2 times. Theresidue was finally dissolved in 2 ml of 0.2% trifluoroacetic acid(TFA). The trifluoroacetic acid-soluble material was applied in 100 μlaliquots to a Poros R2 column (4.6 mm×100 mm) running at 5 ml/min. witha starting buffer of 98% 0.1% trifluoroacetic acid in water/2% 0.1% TFAin acetonitrile. The hydroxyproline-containing protein was eluted withof gradient of 2% 0.1% TFA/acetonitrile to 40% 0.1% TFA/acetonitrileover 25 column volumes (FIG. 62A). The collagen-mimetic eluted between18 and 23% 0.1% TFA/acetonitrile. FIG. 62A is a chromatogram of theelution of hydroxyproline containing CM4 from a Poros RP2 column(available from Perseptive Biosystems, Framingham, Mass.). The arrowindicates the peak containing hydroxyproline containing CM4. Fractionswere assayed by SDS-PAGE and collagen mimetic-containing fractions werepooled and lyophilized. Lyophilized material was stored at −20° C.

EXAMPLE 20 Purification of Proline-Containing Collagen-Like Peptide fromE. coli

The cell pellet from a 500 ml fermentation culture prepared as describedin Example 18 above, was resuspended in 20 ml of Dulbecco's phosphatebuffered saline (pH 7.1) (PBS) containing 10 mM EDTA, 100 μM PMSF, 0.5μg/ml E64, and 0.06 μg/ml aprotinin. Lysozyme (2 mg) was added and thesuspension incubated at 4° C. for 60 minutes. The suspension wassonicated for 5×60 seconds (microtip, power=3.5, Heat Systems XL-2020model sonicator). The sonicated suspension was centrifuged at 20,000×gfor 15 minutes. The supernatent was adjusted to 1% Triton X100 andincubated for 30 minutes at room temperature with 7 ml of glutathionesepharose 4B pre-equilibrated in PBS. The suspension was centrifuged at500 rpm for 3 minutes. The supernatent decanted, and the resin washed 3times with 8 ml of PBS. Bound proteins were eluted with 3 aliquots (2 mleach, 10 minutes gentle rocking at room temperature) of 10 mMglutathione in 50 mM Tris (pH 8.0). Eluants were combined and dialyzed(10,000 MWCO) against 3×4 L of distilled water at 4° C. The contents ofthe dialysis tubing were transferred to a 150 ml round bottom flask andlyophilized to dryness. The residue was dissolved in 3 ml of 70% formicacid and 4 mg of cyanogen bromide was added. The flask was flushed oncewith nitrogen evacuated, and allowed to stir for 18 hours at roomtemperature. The contents of the flask were taken to dryness in vacuo atroom temperature, the residue resuspended in 5 ml of distilled water,and evaporated to dryness again. This was repeated 2 times. The residuewas finally dissolved in 2 ml of 0.2% trifluoroacctic acid (TFA). Thetrifluoroacetic acid-soluble material was applied in 100 μl aliquots toa Poros R2 column (4.6 mm×100 mm) running at 5 ml/min. with a startingbuffer of 98% 0.1% trifluoroacetic acid in water/2% 0.1% TFA inacetonitrile. Bound protein was eluted with of gradient of 2% 0.1%TFA/acetonitrile to 40% 0.1% TFA/acetonitrile over 25 column volumes(FIG. 62B). The collagen-mimetic eluted between 24 and 27% 0.1%TFA/acetonitrile. FIG. 62B is a chromatogram of the elution of prolinecontaining CM4 from a Poros RP2 column. The arrow indicates the peakcontaining proline containing CM4. Fractions were assayed by SDS-PAGEand collagen mimetic-containing fractions were pooled and lyophilized.Lyophilized material was stored at −₂₀° C.

EXAMPLE 21 Amino Acid Analysis of Hydroxyproline-Containing CollagenMimetic and Proline-Containing Collagen Mimetic

Approximately 30 μg of purified hydroxyproline-containing collagenmimetic and proline-containing collagen mimetic prepared as described inExamples 19 and 20, respectively, were dissolved in 250 μl of 6Nhydrochloric acid in glass ampules. The ampules were flushed two timeswith nitrogen, sealed under vacuum, and incubated at 110° C. for 23hours. Following hydrolysis, samples were removed from the ampules andtaken to dryness in vacuo. The samples were dissolved in 15 μl of 0.1Nhydrochloric acid and subjected to amino acid analysis on a HewlettPackard AminoQuant 1090 amino acid analyzer utilizing standard OPA andFMOC derivitization chemistry. Examples of the results of the amino acidanalysis that illustrate the region of the chromatograms where thesecondary amino acids (proline and hydroxyproline) elute are shown inFIGS. 63A through 63D. These Figures also show chromatograms of prolineand hydroxyproline amino acid standards. More particularly, FIG. 63A,depicts a chromatogram of a proline amino acid standard (250 pmol).*indicates a contaminating peak; FIG. 63B depicts a chromatogram of ahydroxyproline amino acid standard (250 pool). *indicates acontaminating peak. FIG. 63C depicts an amino analysis chromatogram ofthe hydrolysis of proline-containing CM4. Only the region of thechromatogram where proline and hydroxyproline elute is shown. *indicatesa contaminating peak. FIG. 63D depicts an amino acid analysischromatogram of the hydrolysis of hydroxyproline-containing CM4. Onlythe region of the chromatogram where proline and hydroxyproline elute isshown. *indicates a contaminating peak.

EXAMPLE 22 Determination of Proline Starvation Conditions for E. coli(Strain JM109 (F−))

A plasmid (pGST-CM4, FIG. 60) containing the gene for collagen mimetic 4(CM4, FIG. 61) genetically linked to the 3′ end of the gene for S.japonicum glutathione S-transferase was used to transform byelectroporation proline auxotrophic E. coli strain JM109 (F—).Transformation cultures were plated on LB agar containing 100 μg/mlampicillin. After overnight incubation at 37° C., a single colony from afresh transformation plate was used to inoculate 2 ml of M9 media (1×M9salts, 0.5% glucose, 1 MM MgCl₂, 0.01% thiamine, 200 μg/ml glycine, 200μg/ml alanine, 100 μg/ml of the other amino acids except proline, and200 μg/ml carbenicillin) and containing 20 μg/ml proline. After growthat 37° C. with shaking for 8 hours, 1.5 ml was used to inoculate 27 mlof M9 media containing 45 μg/ml proline. After incubation at 37° C. withshaking for 7 hours, the culture was centrifuged, the cell pellet washedwith 7 ml of M9 media with no proline, and finally resuspended in 17 mlof M9 media with no proline. This culture was used to inoculate four 35ml cultures of M9 media containing 4 μg/ml proline at an OD600 of 0.028.Cultures were incubated with shaking at 37° C. and the OD600 monitored.After 13.5 hours growth, the OD600 had plateaued. At this time, oneculture was supplemented with proline at 15 μg/ml, one withhydroxyproline at 15 μg/ml, one with all of the amino acids at 15 μg/mlexcept proline and hydroxyproline, and one culture with nothing.Incubation was continued and the OD600 monitored for a total of 24hours. FIG. 64 is a graph of OD600 vs. time for cultures of JM109 (F—)grown to plateau and then supplemented with various amino acids. Thepoint at which the cultures were supplemented is indicated with anarrow. Proline starvation is evident since only the culture supplementedwith proline continued to grow past plateau.

EXAMPLE 23 Hydroxyproline Incorporation Into Type I (α1) Collagen in E.coli

A plasmid (pHuCol(α1)^(Ec), FIG. 65) containing the gene for Type I (α1)collagen with optimized E. coli codon usage (FIG. 39A-39E) (SEQ. ID. NO.19) under control of the tac promoter and containing the gene forchloramnphenicol resistance was used to transform by electroporationproline auxotrophic E. coli strain JM109 (F—). Transformation cultureswere plated on LB agar containing 20 μg/ml chloramphenicol. Afterovernight incubation at 37° C., a single colony from a freshtransformation plate was used to inoculate 100 ml of LB media containing20 μg/ml chloramphenicol. This culture was grown to an OD600m of 0.5 and100 μt aliquots transferred to 1.5 ml tubes. The tubes were stored at−80° C. For expression, a tube was thawed on ice and used to inoculate25 ml of LB media containing 20 μg/ml chloramphenicol. After overnightgrowth at 37° C., a four ml aliquot was withdrawn, centrifuged, the cellpellet washed once with 1 ml of 2×YT media containing 20 μg/mlchloramphenicol, and the washed cells used to inoculate 1 L of 2×YTmedium containing 20 μg/ml chloramphenicol. This culture was grown at37° C. to an OD600nm of 0.8. The culture was centrifuged and the cellpellet washed once with 100 ml of M9 medium (1×M9 salts, 0.5% glucose, 1MM MgCl₂, 0.01% thiamine, 200 μg/ml glycine, 200 μg/ml alanine, 100μg/ml of the other amino acids except proline, and 20 μg/mlchloramphenicol). The cells were resuspended in 910 ml of M9 medium(1×M9 salts, 0.5% glucose, 1 mM MgCl₂, 0.01% thiamine, 200 μg/mlglycine, 200 μg/ml alanine, 100 μg/ml of the other amino acids exceptproline, and 20 μg/ml chloramphenicol) and allowed to grow at 37° C. for30 minutes. NaCl (80 ml of 5 M), hydroxyproline (7.5 ml of 2M), and IPTG(500 μl of 1 M) were added and growth continued for 3 hours. Cells wereharvested by centrifugation and stored at −20° C.

EXAMPLE 24 Hydroxyproline Incorporation Into Type I (α2) in E. coli

A plasmid (pHuCol(α2)^(Ec), FIG. 66) containing the gene for Type I (α2)collagen with optimized E. coli codon usage (FIG. 50A-50E) (SEQ. ID. NO.31) under control of the tac promoter and containing the gene forchloramphenicol resistance was used to transform by electroporationproline auxotrophic E. coli strain JM109 (F—). Transformation cultureswere plated on LB agar containing 20 μg/ml chloramphenicol. Afterovernight incubation at 37° C., a single colony from a freshtransformation plate was used to inoculate 100 ml of LB media containing20 μg/ml chloramphenicol. This culture was grown to an OD600nm of 0.5and 100 μl aliquots transferred to 1.5 ml tubes. The tubes were storedat −80° C. For expression, a tube was thawed on ice and used toinoculate 25 ml of LB media containing 20 μg/ml chloramphenicol. Afterovernight growth at 37° C., a four ml aliquot was withdrawn,centrifuged, the cell pellet washed once with 1 ml of 2×YT mediacontaining 20 μg/ml chloramphenicol, and the washed cells used toinoculate 1 L of 2×YT medium containing 20 μg/ml chloramphenicol. Thisculture was grown at 37° C. to an OD600 nm of 0.8. The culture wascentrifuged and the cell pellet washed once with 100 ml of M9 medium(1×M9 salts, 0.5% glucose, 1 mM MgCl₂, 0.01% thiamine, 200 μg/mlglycine, 200 μg/ml alanine, 100 μg/ml of the other amino acids exceptproline, and 20 μg/ml chloramphenicol). The cells were resuspended in910 ml of M9 medium (1×M9 salts, 0.5% glucose, 1 MM MgCl₂, 0.01%thiamine, 200 μg/ml glycine, 200 μg/ml alanine, 100 μg/ml of the otheramino acids except proline, and 20 μg/ml chloramphenicol) and allowed togrow at 37° C. for 30 minutes. NaCl (80 ml of 5 M), hydroxyproline (7.5ml of 2M), and IPTG (500 μl of 1 M) were added and growth continued for3 hours. Cells were harvested by centrifugation and stored at −20° C.

EXAMPLE 25 Hydroxyproline Incorporation into a C-terminal Fragment ofType I (α1) Collagen in E. coli

A plasmid (pD4-α1, FIG. 67) encoding the gene for the carboxy terminal219 amino acids of human Type I (α1) collagen with optimized E. colicodon usage fused to the 3′-end of the gene for glutathioneS-transferase and under control of the tac promoter and containing thegene for ampicillin resistance was used to transform by electroporationproline auxotrophic E. coli strain JM109 (F—). Transformation cultureswere plated on LB agar containing 100 μg/ml ampicillin. After overnightincubation at 37° C., a single colony from a fresh transformation platewas used to inoculate 100 ml of LB media containing 100 μg/mlampicillin. This culture was grown to an OD600 nm of 0.5 and 100 μlaliquots transferred to 1.5 ml tubes. The tubes were stored at −80° C.For expression, a tube was thawed on ice and used to inoculate 25 ml ofLB media containing 400 μg/ml ampicillin. After overnight growth at 37°C., a four ml aliquot was withdrawn, centrifuged, the cell pellet washedonce with, 1 ml of 2×YT media containing 400 μg/ml ampicillin, and thewashed cells used to inoculate 1 L of 2×YT medium containing 400 μg/mlampicillin. This culture was grown at 37° C. to an OD600 nm of 0.8. Theculture was centrifuged and the cell pellet washed once with 100 ml ofM9 medium (1×M9 salts, 0.5% glucose, 1 mM MgCl₂, 0.01% thiamine, 200μg/ml glycine, 200 μg/ml alanine, 100 μg/ml of the other amino acidsexcept proline, and 400 μg/ml ampicillin). The cells were resuspended in910 ml of M9 medium (1×M9 salts, 0.5% glucose, 1 MM MgCl₂, 0.01%thiamine, 200 μg/ml glycine, 200 μg/ml alanine, 100 μg/ml of the otheramino acids except proline, and 400 μg/ml ampicillin) and allowed togrow at 37° C. for 30 minutes. NaCl (80 ml of 5 M), hydroxyproline (7.5ml of 2M), and IPTG (500 μl of 1 M) were added and growth continued for3 hours. Cells were harvested by centrifugation and stored at −20° C.

EXAMPLE 26 Hydroxyproline Incorporation Into a C-terminal Fragment ofType I (α2) Collagen in E. coli

A plasmid (pD4-α2, FIG. 68) encoding the gene for the carboxy terminal219 amino acids of human Type I (α2) collagen with optimized E. colicodon usage as constructed in accordance with Example 14A fused to the3′-end of the gene for glutathione S-transferase and under control ofthe tac promoter and containing the gene for ampicillin resistance wasused to transform by electroporation proline auxotrophic E. coli strainJM109 (F—). Transformation cultures were plated on LB agar containing100 μg/ml ampicillin. After overnight incubation at 37° C., a singlecolony from a fresh transformation plate was used to inoculate 100 ml ofLB media containing 100 μg/ml ampicillin. This culture was grown to anOD600 nm of 0.5 and 100 μl aliquots transferred to 1.5 ml tubes. Thetubes were stored at −80° C. For expression, a tube was thawed on iceand used to inoculate 25 ml of LB media containing 400 μg/ml ampicillin.After overnight growth at 37° C., a four ml aliquot was withdrawn,centrifuged, the cell pellet washed once with 1 ml of 2×YT mediacontaining 400 μg/ml ampicillin, and the washed cells used to inoculate1 L of 2×YT medium containing 400 μg/ml ampicillin. This culture wasgrown at 37° C. to an OD600 nm of 0.8. The culture was centrifuged andthe cell pellet washed once with 100 ml of M9 medium (1×M9 salts, 0.5%glucose, 1 MM MgCl₂, 0.01% thiamine, 200 μg/ml glycine, 200 μg/mlalanine, 100 μg/ml of the other amino acids except proline, and 400μg/ml ampicillin). The cells were resuspended in 910 ml of M9 medium(1×M9 salts, 0.5% glucose, 1 mM MgCl₂, 0.01% thiamine, 200 μg/mlglycine, 200 μg/ml alanine, 100 μg/ml of the other amino acids exceptproline, and 400 μg/ml ampicillin) and allowed to grow at 37° C. for 30minutes. NaCl (80 ml of 5 M), hydroxyproline (7.5 ml of 2M), and IPTG(500 μl of 1 M) were added and growth continued for 3 hours. Cells wereharvested by centrifugation and stored at −20° C.

EXAMPLE 27 Purification of Hydroxyproline-Containing C-terminal Fragmentof Type I (α1) Collagen

Cell paste harvested from a 1 L culture grown as in Example 25 wasresuspended in 30 ml of lysis buffer (2M urea, 137 mM NaCl, 2.7 mM KCl,4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, 10 mM EDTA, 10 mM PME, 0.1% Triton X-100,pH 7.4) at 4° C. Lysozyme (chicken egg white) was added to 100 μg/ml andthe solution incubated at 4° C. for 30 minutes. The solution was passedtwice through a cell disruption press (SLM Instruments, Rochester, N.Y.)and then centrifuged at 30,000×g for 30 minutes. The pellet wasresuspended in 30 ml of 50 mM Tris-HCl, pH 7.6, centrifuged at 30,000×gfor 30 minutes, and the pellet solubilized in 25 ml of solubilizationbuffer (8M urea, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄,5 mM EDTA, 5 mM PME). The solution was centrifuged at 30,000×g for 30minutes and supernatent dialyzed against two changes of 4 L of distilledwater at 4° C. Following dialysis, the entire mixture was lyophilized.The lyophilized solid was dissolved in 0.1M HCl in a flask withstirring. After addition of a 5-fold excess of crystalline BrCN, theflask was evacuated and filled with nitrogen. Cleavage was allowed toproceed for 24 hrs, at which time the solvent was removed in vacuo. Theresidue was dissolved in 0.1% trifluoroacetic acid (TFA) and purified byreverse-phase HPLC using a Vydac C4 RP-HPLC column (10×250 mm, 5 μ, 300Å) on a BioCad Sprint system (Perceptive Biosystems, Framingham, Mass.).Hydroxyproline-containing D4 protein was eluted with a gradient of15-40% acetonitrile/0.1% TFA over a 45 minute period. Protein D4-α1eluted at 26% acetonitrile/0.1% TFA.

EXAMPLE 28 Purification of Hydroxyproline-Containing C-terminal Fragmentof Type I (α2) Collagen

Cell paste harvested from a 1 L culture grown as in Example 26 wasresuspended in 30 ml of lysis buffer (2M urea, 137 mM NaCl, 2.7 mM KCl,4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, 10 mM EDTA, 10 mM PME, 0.1% Triton X-100,pH 7.4) at 4° C. Lysozyme (chicken egg white) was added to 100 μg/ml andthe solution incubated at 4° C. for 30 minutes. The solution was passedtwice through a cell disruption press (SLM Instruments, Rochester,N.Y.). and then centrifuged at 30,000×g for 30 minutes. The pellet wasresuspended in 30 ml of 50 mM Tris-HCl, pH 7.6, centrifuged at 30,000×gfor 30 minutes, and the pellet solubilized in 25 ml of solubilizationbuffer (8M urea, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄,5 mM EDTA, 5 mM PME). The solution was centrifuged at 30,000×g for 30minutes and supernatent dialyzed against two changes of 4 L of distilledwater at 4° C. Following, dialysis, the entire mixture was lyophilized.The lyophilized solid was dissolved in 0.1M HCl in a flask withstirring. After addition of a 5-fold excess of crystalline BrCN, theflask was evacuated and filled with nitrogen. Cleavage was allowed toproceed for 24 hrs, at which time the solvent was removed in vacuo. Theresidue was dissolved in 0.1% trifluoroacetic acid (TFA) and purified byreverse-phase HPLC using a Vydac C4 RP-HPLC column (10×250mm, 5μ, 300 Å)on a BioCad Sprint system (Perceptive Biosystems, Framingham, Mass.).Hydroxyproline-containing D4 protein was eluted with a gradient of15-40% acetonitrile/0.1% TFA over a 45 minute period. Protein D4-α2eluted at 25% acetonitrile/0.1% TFA.

EXAMPLE 29 Amino Acid Composition Analysis of Hydroxyproline-ContainingC-terminal Fragment of Type I (α1) Collagen

Protein D4-α1 (10 μg) purified as in Example 27 was taken to dryness invacuo in a 1.5 ml microcentrifuge tube. A sample was subjected to aminoacid analysis at the W.M. Keck Foundation Biotechnology ResourceLaboratory (New Haven, Conn.) on an Applied Biosystems sequencerequipped with an on-line HPLC system. The experimentally determinedsequence of the first 13 amino acids (SEQ. ID. NO. 41) and the sequencepredicted from the DNA sequence (SEQ. ID. NO. 42) are shown in FIG. 69.A sample of protein D4-α1 was subjected to mass spectral analysis on aVG Biotech BIO-Q quadrople analyzer at M-Scan, Inc. (West Chester, Pa.).The mass spectrum and the predicted molecular weight of protein D4-α1 ifit contained 100% hydroxyproline in lieu of proline are given in FIG.70. The predicted molecular weight of protein D4-α1 containing 100%hydroxyproline in lieu of proline is 20807.8 Da. The experimentallydetermined molecular weight was 20807.5 Da.

EXAMPLE 30 Construction of Carboxy Terminal 219 Amino Acids of HumanCollagen Type I (α1) Fragment Gene with Optimized E. Coli Codon Usage.

The nucleotide sequence of the 657 nucleotide gene for the carboxyterminal 219 amino acids of human Type I (α1) collagen with optimized E.Coli codon usage is shown in FIG. 71. For synthesis of this gene, uniquerestriction sites C) were identified or created approximately every 150base pairs. Oligos of approximately 80 nucleotides were synthesized on aBeckman Oligo 1000 DNA synthesizer, cleaved and deprotected with aqueousNH₄OH, and purified by electrophoresis in 7M urea/12% polyacrylamidegels. Each set of oligos was designed to have an EcoR I restrictionenzyme site at the 5′ end, a unique restriction site near the 3′ end,followed by the TAAT stop sequence and a Hind III restriction enzymesite at the very 3′ end. The first four oligos, comprising the first 84amino acids of the carboxy terminal 219 amino acids of human Type I (α1)collagen with optimized E. coli codon usage, are given in FIG. 81 (SEQ.ID. NOS. 47-50).

Oligos N4-1 (SEQ. ID. NO. 47) and N4-2 (SEQ. ID. NO. 48) (1 μg each)were annealed in 20 μL of T7 DNA polymerase buffer (40 mM Tris-HCl (pH8.0), 5 mM MgCl₂, 5 mM dithiothreitol, 50 mM NaCl, 0.05 mg/mL bovineserum albumin) by heating at 90° C. for 5 minutes followed by slowcooling to room temperature. After brief centrifugation at 14,000 rpm,10 units of T7 DNA polymase and 2 μL of a solution of all four dNTPs(DATP, dGTP, dCTP, dTTP, 2.5 mM each) were added to the annealed oligos.Extension reactions were incubated at 37° C. for 30 minutes and thenheated at 70° C. for 10 minutes. After cooling to room temperature, HindIII buffer (5 μL of 10× concentration), 20 μL of H₂O, and 10 units ofHind III restriction enzyme were added and the tubes incubated at 37° C.for 10 hours. Hind III buffer (2 μL of 10× concentration), 13.5 μL of0.5M Tris HCl (pH 7.5), 1.8 μL of 1% Triton X100, 5.6 μL of H₂O, and 20U of EcoR I were added to each tube and incubation continued for 2 hoursat 37° C. Digests were extracted once with an equal volume of phenol,once with phenol/chloroform/isoamyl alcohol, and once withchloroform/isoamyl alcohol. After ethanol precipitation, the pellet wasresuspended in 10 μL of TE buffer (10 mM Tris HCl (pH 8.0), 1 mM EDTA).Resuspended pellet 4 μL of was ligated overnight at 16° C. with agarosegel-purified EcoRI/Hind III digested pBSKS⁺ vector (1 μg) using T4 DNAligase (100 units). One half of the transformation mixture wastransformed by heat shock into DH5α cells and 100 μL of the 1.0 mLtransformation mixture was plated on Luria Broth (LB) agar platescontaining 70 μg/mL ampicillin. Plates were incubated overnight at 37°C. Ampicillin resistant colonies (6-12) were picked and grown overnightin LB media containing 70 μg/mL ampicillin. Plasmid DNA was isolatedfrom each culture by Wizard Minipreps (Promega Corporation, MadisonWis.) and screened for the presence of the approximately 120 base pairinsert by digestion with EcoRI and Hind III and running the digestionproducts on agarose electrophoresis gels. Clones with inserts wereconfirmed by standard dideoxy termination DNA sequencing. The correctclone was named pBSN4-1.

Oligos N4-3 (SEQ. ID. NO. 49) and N4-4 (SEQ. ID. NO. 50) (FIG. 81) weresynthesized, purified, annealed, extended, and cloned into pBSKS⁺following exactly the same procedure given above for oligos N4-1 andN4-2. The resulting plasmid was named pBSN4-2A. To clone together thesections of the collagen gene from pBSN4-1 and pBSN4-2A, plasmid pBSN4-1(1 μg) was digested for 2 hours at 37° C. with Apa L1 and Hind III. Thedigested vector was purified by agarose gel electrophoresis. PlasmidpBSN4-2A (3 μg) was digested for 2 hours at 37° C. with Apa L1 and HindIII and the insert purified by agarose gel electrophoresis. Apa L1/HindIII digested pBSN4-1 was ligated with this insert overnight at 16° C.with T4 DNA ligase. One half of the ligation mixture was transformedinto DH5α cells and 1/10 of the transformation mixture was plated on LBagar plates containing 70 μg/mL ampicillin. After overnight incubationat 37° C., ampicillin-resistant clones were picked and screened for thepresence of insert DNA as described above. Clones were confirmed bydideoxy termination sequencing. The correct clone was named pBSN4-2.

In a similar manner, the remainder of the gene for the carboxy terminal219 amino acids of human Type I (α1) collagen with optimized E. colicodon usage was constructed such that the final DNA sequence is thatgiven in FIG. 71 (SEQ. ID. NO. 43).

It will be understood that various modifications may be made to theembodiments disclosed herein. For example, it is contemplated that anyprotein produced by prokaryotes and eukaryotes can be made toincorporate one or more amino acid analogs in accordance with thepresent disclosure. Therefore, the above description should not beconstrued as limiting, but merely as exemplifications of preferredembodiments. Those skilled in art will envision other modificationswithin the scope and spirit of the claims appended hereto.

1. (canceled)
 2. (canceled)
 3. Nucleic acid encoding a humanExtracellular Matrix Protein (EMP) or fragment thereof wherein the codonusage in the nucleic acid sequence reflects preferred codon usage in aprokaryotic cell.
 4. Nucleic acid according to claim 3 wherein theprokaryotic cell is E. coli.
 5. Nucleic acid according to claim 3wherein the EMP is selected from the group consisting of collagen,fibrinogen, fibronectin and collagen-like peptide.